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Article

Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages

by
Riley J. Morrow
,
Amr H. Allam
,
Josh Konecnik
,
David Baloyan
,
Christine Dijkstra
,
Moritz F. Eissmann
,
Saumya P. Jacob
,
Megan O’Brien
,
Ashleigh R. Poh
*,† and
Matthias Ernst
*,†
The Olivia Newton-John Cancer Research Institute and School of Cancer Medicine, La Trobe University, Heidelberg, VIC 3084, Australia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cells 2022, 11(24), 4104; https://doi.org/10.3390/cells11244104
Submission received: 17 November 2022 / Revised: 6 December 2022 / Accepted: 15 December 2022 / Published: 17 December 2022
(This article belongs to the Special Issue MYC Signaling in Cancer)
Browse Figures
Versions Notes

Abstract

:
Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysMCre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages.

1. Introduction

Macrophages are a major component of the tumor microenvironment and are associated with a poor prognosis in most solid malignancies. Depending on their activation status, macrophages can exert dual influences on tumorigenesis by either enhancing immune cell activation or by antagonizing cytotoxic immune responses. Classically activated M1 macrophages are important drivers of anti-tumor immunity by mediating antibody-dependent cellular cytotoxicity and phagocytosis, amplifying innate and adaptive immune surveillance, and promoting tumor necrosis [1]. In contrast, alternatively activated M2 macrophages play a pivotal role in tumor initiation and progression by contributing to angiogenesis, immune suppression, and extracellular matrix remodeling, as well as resistance to chemotherapy and immune checkpoint inhibitors [2]. For this reason, therapies aimed at reprogramming tumor-associated macrophages (TAMs) away from an alternatively activated endotype represent a promising strategy for anti-cancer therapy.
The oncoprotein c-Myc (Myc) is the prototypical member of the Myc family of transcription factors that regulate a broad spectrum of biological processes, including proliferation, angiogenesis, tissue remodeling, metabolism, and hematopoiesis [3,4,5]. Although Myc activity is tightly regulated in normal cells, deregulation of Myc is observed in up to 70% of tumors [6] and is associated with a poor prognosis and reduced patient survival [7]. In support of its tumor cell-intrinsic role, the inactivation of Myc in cancer cells results in tumor regression by promoting proliferative arrest, cellular senescence, and the induction of apoptosis [8]. Meanwhile, Myc also plays a major tumor cell-extrinsic role by facilitating immune evasion through decreased expression of MHC I and upregulation of inhibitory cytokines and immune checkpoint proteins [9,10,11]. Notably, Myc has been suggested to transcriptionally regulate the expression of genes associated with alternative macrophage polarization [12,13,14], while the inhibition of Myc skews macrophages towards a classically activated inflammatory endotype [15]. These findings suggest that inhibition of Myc in TAMs may offer a therapeutic opportunity to curb tumor growth by reprogramming the immunosuppressive tumor microenvironment conferred by alternatively activated macrophages.
In this study, we explored as a first proof-of-principle assessment whether conditional ablation of Myc in host macrophages impairs tumor growth by reducing the polarization of alternatively activated TAMs in vivo. Unexpectedly, we found no evidence that conditional ablation of Myc expression in macrophages reduces tumor growth across four complementary pre-clinical models. Our observations suggest that systemic targeting of Myc activity may therefore confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than suppressing Myc in TAMs.

2. Materials and Methods

2.1. Mice

Age- and sex-matched mice were bred and maintained in specific pathogen-free facilities at La Trobe University and the Austin Hospital, Australia. The LysMCre/+ strain [16] was crossed with the Mycfl/fl strain [17] to generate LysMCre/+;Mycfl/fl and LysM+/+;Mycfl/fl mice [14]. Where indicated, LysMCre/+;Mycfl/fl and LysM+/+;Mycfl/fl mice were additionally crossed with Gp130F/F mice [18] to generate LysMCre/+;Mycfl/fl;Gp130F/F and LysM+/+;Mycfl/fl;Gp130F/F animals. The Tff1CreERT2 strain [19] was crossed with Mycfl/fl;Gp130F/F animals to generate Tff1CreERT2;Mycfl/fl;Gp130F/F compound mutant mice. All animal studies were approved and conducted in accordance with the Animal Ethics Committee at La Trobe University and the Olivia Newton-John Cancer Research Institute/Austin Hospital.

2.2. Tumor Models

The mouse MC38 colon cancer and B16F10 melanoma cell lines were maintained in DMEM/F12 (Gibco #11320033, Waltham, MA, USA) supplemented with 10% FCS at 37 °C with 10% CO2. Cell lines tested negative for mycoplasma. Six-week-old LysMCre/+;Mycfl/fl and LysM+/+;Mycfl/fl mice were subcutaneously injected with 1 × 106 MC38 or 2 × 105 B16F10 cells into the right flank. Mice were collected at 2 weeks following tumor cell injection.
Mouse gastric tumor organoids were derived from gastric adenocarcinomas of KrasG12D/+;Pik3caH1047R/+;Trp53R172H/+ (KPT) mutant mice (M.F. Eissmann; unpublished) and maintained as previously described [20,21]. Six-week-old LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl littermates were subcutaneously injected with 900 organoids into the right flank. Mice were collected at 3 weeks following tumor organoid injection.
LysM+/+;Mycfl/fl;Gp130F/F and LysMCre/+;Mycfl/fl;Gp130F/F animals were euthanized at 100 days of age. Stomachs were dissected longitudinally along the greater curvature, and gastric tumors were dissected and weighed.

2.3. Ablation of Gastric Epithelial Cells via Tamoxifen Treatment

Ninety-day-old Tff1CreERT2;Mycfl/fl;Gp130F/F mice were administered a total of 6 mg tamoxifen (dissolved in 10% ethanol and 90% sunflower oil) via intraperitoneal injection (1mg/mL per dose; 2 doses a day over 3 consecutive days). Mice were euthanized 7 days following the last tamoxifen injection. Stomachs were dissected longitudinally along the greater curvature, and gastric tumors were dissected and weighed.

2.4. Immunofluorescence

Paraffin-embedded formalin-fixed sections were dewaxed in xylene and rehydrated in ethanol. Antigen retrieval was performed by incubating slides in EDTA buffer (pH 9) for 20 min at 95 °C. Sections were immersed in 3% H2O2 for 10 min at room temperature to inhibit endogenous peroxidase activity, washed in TBST, then blocked in 2% bovine serum for 1 h at room temperature. Monoplex staining with primary antibodies was performed using the OPAL 7 color kit (Akoya Biosciences, Marlborough, MA, USA) as previously described [22]. Following incubation with HRP-conjugated secondary antibodies, slides were incubated with individual tyramide signal amplification (TSA)-conjugated fluorophores (Akoya Biosciences, Marlborough, MA, USA) for 10 min at room temperature then washed with TBST. Slides were mounted with Fluoromount-G (Thermofisher #00-4958-02, Waltham, MA, USA) and scanned using a 20× objective on the Vectra® 3 automated quantitative pathology imaging system (Akoya Biosciences, Marlborough, MA, USA). inForm software (Version 2.2, Akoya Biosciences, Marlborough, MA, USA) was used to build a spectral library using monoplex scans.
Following the identification of optimal staining parameters for monoplex staining, staining of the full multiplex panel was performed. First, staining for Myc (Abcam #ab32072, Waltham, MA, USA) was performed using the steps outlined above. After incubation with TSA dye, slides were washed in TBST, and antigen retrieval was performed again to remove the primary and secondary antibody complex. Next, F4/80 staining (Cell Signaling #D2S9R, Danvers, MA, USA) was performed. After staining with TSA dye, slides were washed in TBST. Sections were then incubated with spectral DAPI (Akoya Biosciences, Marlborough, MA, USA), washed in dH2O, and mounted with Fluoromount-G. Slides were scanned using a 20× objective on the Vectra® 3 automated quantitative pathology imaging system. inForm software (Version 2.2, Akoya Biosciences, Marlborough, MA, USA) was used to analyze images.

2.5. Flow Cytometry

Tumors were cut into 1 mm pieces and digested in Collagenase/Dispase (Roche #11097113001, Basel, Switzerland) and DNase I (Roche #10104159001, Basel, Switzerland) diluted in Ca2+/Mg2+-free HBSS media (Gibco #14170112, Waltham, MA, USA) plus 10% FCS for 30 min at 37 °C [23]. Samples were vortexed for 15 s, then filtered and washed in PBS plus 10% FCS. After incubation with Fc block (ThermoFisher #14-9161-73, Waltham, MA, USA) on ice for 10 min, cells were stained with fluorophore-conjugated primary antibodies for 20 min on ice in the dark, washed twice, and re-suspended in PBS supplemented with 10% FCS.
The antibodies used included CD45.2 (Clone 30-F11; BioLegend #103116, San Diego, CA, USA), F4/80 (Clone BM8; BioLegend #123114, San Diego, CA, USA), CD11b (Clone M1/70; BioLegend #101208, San Diego, CA, USA), Ly6G (Clone 1A8, BD Biosciences #560602, Franklin Lakes, NJ, USA), Ly6C (Clone HK.4, eBioscience #48-5932-82, Waltham, MA, USA), TCRβ (Clone H57-597; BD Biosciences #553170, Franklin Lakes, NJ, USA) and CD8a (Clone 53-6.7; BioLegend #100712, San Diego, CA, USA).
Flow cytometry was performed on a BD FACS Aria III cell sorter and analyzed using FlowJo software (Version 10, Oregon, USA). Background fluorescence was estimated using isotype controls, fluorescent-minus-one controls, and unstained controls. Dead cells were excluded by Sytox Blue (ThermoFisher #S34857, Waltham, MA, USA) staining.

2.6. RNA Extraction and qPCR

RNA extraction was performed using the RN-easy Micro Plus kit (Qiagen #74034, Hilden, Germany) and RN-easy Mini Plus kit (Qiagen #74134, Hilden, Germany) for FACS-isolated cells and whole tumors, respectively. cDNA from FACS-isolated cells was generated using the SuperScript™ IV First-Strand Synthesis System (ThermoFisher #18091050, Waltham, MA, USA), and cDNA from tumors was generated using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher #4368814, Waltham, MA, USA).
qPCR analysis on each biological sample was performed using technical replicates with Taqman® Real-Time PCR Master mix and probes (ThermoFisher #4352042, Waltham, MA, USA). Samples were run on the Viia7 Real-Time PCR System for 40 cycles (95 °C for 15 s, 60 °C for 1 min) with an initial holding stage (95 °C for 3 min). Fold changes in gene expression were obtained using the 2−ΔΔCT method [24].
The Taqman probes used were mouse 18s (Mm04277571_s1), Gapdh (Mm99999915_g1), Hprt (Mm03024075_m1) Myc (Mm00487804_m1) Il4 (Mm00445259_m1), Il10 (Mm01288386_m1), Il13 (Mm00434204_m1), Tgfβ (Mm01227699_m1), Vegfα (Mm00437306_m1), Arg1 (Mm00475988_m1), Ym1 (Mm00657889_mH), Il1β (Mm00434228_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), GzmB (Mm00442837_m1), and Prf1 (Mm00812512_m1).

2.7. Western Blot Analysis

Tumor protein lysates were resolved on 4–12% NuPAGE Bis-Tris gels [25,26]. Following dry transfer using an iBlot 2 (ThermoFisher, Waltham, MA, USA), PVDF membranes were blocked in Intercept Blocking Buffer (LI-COR Biosciences #927-70001, Lincoln, NE, USA) for 1 h at room temperature. Membranes were incubated overnight in anti-Myc antibody (Abcam #ab32072, Waltham, MA, USA) and anti-actin antibody (loading control; Sigma #A228, Saint Louis, MO, USA) at 4 °C. The next day, blots were washed twice in TBST, then stained with fluorescent-conjugated secondary antibodies (LI-COR Biosciences #926-32221 and #926-32210, Lincoln, NE, USA) for 1 h at room temperature. After two additional washes in TBST, signals were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

2.8. Isolation of Bone-Marrow-Derived Macrophages

Bone marrow was harvested from the femur and tibia of mice by flushing with sterile PBS as previously described [25,26]. Cells were washed twice in PBS and filtered through a 70 μm sieve. The single-cell suspension was then cultured in Macrophage Media (DMEM/F12 supplemented with 10% FCS and 20% L929 conditioned media). To fully differentiate bone-marrow-derived macrophages, cells were cultured for 7 days with fresh media changed every 3 days. Adherent macrophages were detached from plates using a cell scraper and processed for downstream analysis.

2.9. Isolation of Peritoneal Macrophages

The peritoneal cavity of mice was flushed with 5 mls of PBS supplemented with 3% FCS and gently massaged to detach immune cells. The cell suspension was aspirated and pelleted by centrifugation for downstream FACS sorting to isolate CD45+CD11b+F4/80+Ly6C−LygG− macrophages.

2.10. Isolation of Splenic Macrophages

Spleens were mashed through a 70 µm filter, resuspended in 30 mls of PBS, and pelleted by centrifugation. Cells were incubated in red cell lysis buffer for 5 min, washed in PBS plus 10% FCS, and pelleted by centrifugation for downstream FACS sorting to isolate CD45+CD11b+F4/80+Ly6C−LygG− macrophages.

2.11. Quantification and Statistical Analysis

All experiments were performed at least twice with a minimum of three age- and sex-matched mice per group. The specific n (number of animals) used per cohort is indicated in the respective figure legends and shown as individual data points. No data were excluded from the analysis. Tumor weights were recorded by an independent assessor who was blinded to the experimental conditions. Statistical analysis was conducted using GraphPad Prism Software (Version 8). Comparisons between two mean values were performed with a 2-tailed Student’s t-test. A p value of less than 0.05 was considered statistically significant.

3. Results

3.1. Myc Expression Is Reduced in Macrophages of LysMCre/+;Mycfl/fl Mice

The transgenic LysMCre/+ knock-in strain enables conditional depletion of Myc gene expression in macrophages of Mycfl/fl mice due to lox(p) sites flanking exons 2 and 3 of Myc [27]. Consequently, the abundance of functional Myc protein in mature macrophages of LysMCre/+;Mycfl/fl mice is reduced by up to 90% [14]. To validate these observations, we performed qPCR analysis on peritoneal and bone-marrow-derived macrophages isolated from LysMCre/+;Mycfl/fl mice. We observed an 80–90% reduction in Myc expression in these cells compared to cells purified from LysMCre transgene-deficient LysM+/+;Mycfl/fl littermate controls (Figure 1). Consistent with previous observations of limited Cre recombinase activity conferred by LysMCre in splenic myeloid cells [27], we confirmed a lack of quantitative Myc ablation in these cells (Figure 1).

3.2. Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages

To assess the contribution of Myc signaling in macrophages to the growth of tumors with low immunogenicity, we crossed LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl mice to the Gp130F/F mouse model. In Gp130F/F mice, a disruption of the Socs3-dependent negative feedback loop on the shared IL-6 cytokine family gp130 receptor subunit results in excessive STAT3 signaling that promotes the spontaneous development of gastric adenomas from 6 weeks of age [28,29]. Owing to the ontogenetic relationship between TAMs and bone-marrow-derived macrophages, we first confirmed reduced Myc expression in tumors and TAMs of LysM+/+;Mycfl/fl;Gp130F/F mice (Figure 2A and Supplementary Figure S1A,B). However, we did not observe a difference in tumor burden between LysM+/+;Mycfl/fl;Gp130F/F and LysMCre/+;Mycfl/fl;Gp130F/F littermates (Figure 2B). By contrast, tamoxifen-induced Cre-mediated ablation of Myc in the gastric epithelium of tumor-bearing Tff1CreERT2;Mycfl/fl;Gp130F/F compound mutant mice significantly reduced gastric tumor burden (Supplementary Figure S1C,D).
Because our observations suggested that Myc expression in TAMs may not affect tumor growth during early adenomatous stages, we next determined whether Myc ablation in TAMs affected the growth of tumors that develop into invasive carcinomas. For this, we established subcutaneous tumors in LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts using tumor organoids derived from invasive gastric adenocarcinomas of KrasG12D/+;Pik3caH1047R/+;Trp53R172H/+ (KPT) mice. We confirmed reduced Myc expression in KPT tumors excised from LysMCre/+;Mycfl/fl hosts; however, these tumors were of a comparable size to those collected from Myc-proficient LysM+/+;Mycfl/fl hosts (Figure 2C,D).
To ascertain whether our observations remained pertinent to gastric tumors, we next assessed the contribution of myeloid cell-specific Myc depletion in hosts engrafted with highly immunogenic B16F10 melanoma or MC38 colon cancer allografts. Despite an 80% reduction in Myc expression in LysMCre/+;Mycfl/fl TAMs, we could not detect differences in the growth of B16F10 tumors between LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts (Figure 2E,F). In contrast, reduced Myc expression in MC38 TAMs coincided with larger tumors in LysMCre/+;Mycfl/fl hosts compared to their LysM+/+;Mycfl/fl littermates (Figure 2G,H). Collectively, our results suggest that Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade.

3.3. Conditional Ablation of Myc in TAMs Neither Reduces Tumor Immune Suppression Nor Impairs Alternative Macrophage Polarization

Myc expression in TAMs has been shown to regulate tumor growth by reinforcing an immunosuppressive microenvironment [13,14,30], while the inhibition of Myc enables T-cell-mediated immune surveillance [31,32]. We therefore profiled tumors from Gp130F/F, KPT, B16F10, or MC38 models that arose in either LysM+/+;Mycfl/fl or LysMCre/+;Mycfl/fl hosts for markers associated with immune suppression (i.e., Il4, Il10, Il13, Arg1, Ym1, Mrc1, Tgfβ) and immune activation (i.e., Il1β, Il12, Tnfα, Nos2, Ifnγ, GzmB and Prf1). Assessment of the corresponding transcripts by qPCR across all four models showed comparable gene expression levels in tumors irrespective of the genotype of the hosts (Figure 3A–D).
To investigate whether in vivo depletion of Myc in macrophages resulted in more subtle changes in TAM and CD8+ T-cell recruitment and activation, we performed flow cytometry to quantify the proportion of these immune cells in B16F10 and MC38 tumors established in LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. We observed a comparable abundance of TAMs and CD8+ T-cells in B16F10 tumors between LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts (Figure 4A). In line with the increased tumor burden observed with MC38 tumor cells in LysMCre/+;Mycfl/fl hosts, we also observed an increased proportion of TAMs in these tumors compared to LysM+/+;Mycfl/fl controls. However, this difference did not impact the abundance of tumor-infiltrating CD8+ T-cells, which remained similar across both groups (Figure 4B).
We next purified TAMs from subcutaneous B16F10 and MC38 tumors established in LysMCre/+;Mycfl/fl hosts to determine whether their endotype would differ from TAMs associated with tumors from LysM+/+;Mycfl/fl hosts. We assessed gene signatures comprising prototypical markers for classical (i.e., Il1β, Tnfα, Nos2) and alternative macrophage activation (i.e., Il4, Il10, Il13, Tgfβ, Vegfα, Arg1, Ym1). We did not detect significant differences for any of these markers between TAMs isolated from B16F10 tumors of LysMCre/+;Mycfl/fl and LysM+/+;Mycfl/fl hosts (Figure 4C). By contrast, we observed increased expression of genes associated with alternative macrophage polarization (e.g., Vegfα, Arg1, Ym1) in TAMs isolated from MC38 tumors of LysMCre/+;Mycfl/fl hosts, while the expression of genes associated with classical macrophage polarization remained unchanged (Figure 4D).
To assess whether the increased MC38 tumor burden and alternatively activated endotype of TAMs observed in LysMCre/+;Mycfl/fl hosts could be attributed to reduced activity of CD8+ effector T-cells, we next assessed the expression of cytotoxic molecules (i.e., GzmB and Prf1) in CD8+ T-cells isolated from MC38 tumors of LysMCre/+;Mycfl/fl and LysM+/+;Mycfl/fl hosts (Figure 4D). Surprisingly, we did not observe a difference in GzmB or Prf1 expression between CD8+ T-cells isolated from MC38 tumors between groups, suggesting that impaired T-cell mediated anti-tumor immune responses are unlikely to explain the increased tumor burden observed in LysMCre/+;Mycfl/fl hosts.

4. Discussion

In this study, we provide comprehensive evidence across complementary early adenomatous and carcinoma models that genetic ablation of Myc in TAMs fails to confer increased host-mediated anti-tumor responses. Because the contribution of TAMs to tumorigenesis can be mediated by affecting the recruitment and/or activation of effector T-cells, we also confirmed that Myc-depleted macrophages did not improve host anti-tumor immune responses in highly immunogenic tumors. We surmise from our data that systemic targeting of Myc is unlikely to confer tumor cell-extrinsic therapeutic benefits that arise from direct modulation of TAM polarization.
Macrophages constantly alter their endotype in response to their surroundings, which include signals derived from pathogens and immune cells [2,33,34]. This allows them to orchestrate diverse activities, including stimulating host immunity, removal of cellular debris, and wound healing. Classically activated M1 macrophages are activated following exposure to bacterial lipopolysaccharide or Th1-associated cytokines (e.g., TNFα and IFNγ), and exhibit a pro-inflammatory and phagocytic endotype [35,36]. In contrast, alternatively activated M2 macrophages play a pivotal role in immune suppression, wound healing, and fibrosis, and are induced by parasitic infections, immune-suppressive cytokines (e.g., IL4, IL10, IL13), and glucocorticoids [35,36]. Given the broad range of environmental cues dictating macrophage endotypes, transcription factors including peroxisome proliferation-factor receptors (PPARs) [37], signal transducer and activator of transcription (STAT)s [38,39], CAATT enhancer-binding proteins [40], interferon regulatory factors (IRFs) [41], Kruppel-like factors (KLF) [42], and NF-κB [43] have been shown to functionally polarize macrophages. Moreover, alterations to signaling molecules upstream of these transcription factors, including CSF1R [44], PI3Kγ [45], or the SRC kinase HCK [23,25,26,46], are currently exploited to limit alternative macrophage polarization as adjuvant therapy for cancer.
Myc has been previously implicated as part of the signaling cascade that affects macrophage polarization [47]; however, these observations remain conflicting. For example, the inhibition of Myc suppresses HIF1α, a key metabolic regulator of classical M1 macrophage polarization [48,49,50]. In contrast, degradation of Myc by the E3 ligase FBXW7 restricts alternative M2 TAM polarization and limits tumor progression [51]. In another study, Myc activity enhanced M2 macrophage polarization by transcriptionally repressing miR-26a [52,53]. Finally, Pello and colleagues observed that genetic ablation of Myc using LysMCre/+;Mycfl/fl hosts reduced the growth of subcutaneous B16F10 tumors and correlated these in vivo findings with a bias away from an alternatively activated gene expression signature in TAMs [13,14].
Surprisingly, our observations have failed to substantiate the observation of impaired B16F10 tumors in LysMCre/+;Mycfl/fl hosts. Importantly, we also failed to document increased anti-tumor activity despite less than 20% residual Myc expression in TAMs or tumors of LysMCre/+;Mycfl/fl hosts in the MC38 and KPT allograft models, as well as in the endogenous Gp130F/F adenoma model. Although phenotypic differences have been described across the various conditional Myc alleles [17,54], we note that in their studies Pello and colleagues [13,14] used the same LysMCre/+ transgene (Lyz2tm1(cre)Ifo) [16] and Mycfl/fl allele (Myctm2Fwa) [17] as we used for our assessment. These contrasting findings may be attributed to differences in the genetic background of the mouse models, the microbiome, and other less controllable (e.g., environmental) influences between the different studies. However, the identification of these parameters remains challenging because the exact molecular mechanism by which myeloid-specific Myc ablation affects tumor growth remains unknown over and above a “guilt by association” correlation with the extent of M2 macrophage polarization. Indeed, we were unable to correlate the differences observed between MC38 tumor allografts in LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts with either altered abundance or activity of cytotoxic T-cells, suggesting that the increased tumor burden in LysMCre/+;Mycfl/fl hosts is unlikely to result from reduced adaptive anti-tumor immunity. These observations are in striking contrast to the observation that excessive Myc activity in Kras-driven lung adenomas accelerates adenocarcinoma development as a result of an immune-suppressed stroma. The latter arises from instructive signals by tumor-derived factors (i.e., CCL9 and IL23) that attract TAMs and mediate the exclusion of effector immune cells, respectively [55].
In summary, our collective insights from our models of substantial Cre/lox-mediated ablation of Myc in macrophages suggest that Myc is unlikely to serve as a reliable therapeutic anti-tumor target in these host cells. This is in stark contrast to the reduced tumor burden observed in models of Cre/lox-mediated Myc ablation in tumor cells, including our own observations in Tff1CreERT2;Mycfl/fl;Gp130F/F Myc-depleted gastric adenomas. Thus, we predict that the therapeutic administration of systemic-acting anti-Myc therapies in humans may confer their main therapeutic effect directly on cancer cells, rather than by directly reinvigorating anti-tumor immunity. Indeed, our observations suggest careful assessment, as some tumors may thrive upon the inhibition of Myc in myeloid cells. In-depth comparison between treatment response and cellular analysis in ongoing phase I/II trials with the Myc-antagonist Omomyc/OMO-103 in patients with advanced solid malignancies (NCT04808362) will ultimately identify the mechanisms that underpin the clinical impact of Myc inhibition.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells11244104/s1, Figure S1: Epithelial-specific ablation of Myc reduces gastric tumor growth in Gp130F/F mice.

Author Contributions

Conception and design: R.J.M., A.R.P. and M.E.; development of methodology: R.J.M., A.H.A., J.K., D.B., C.D., M.F.E., S.P.J., M.O., A.R.P. and M.E.; acquisition of data: R.J.M., A.H.A., J.K., D.B., C.D., M.F.E., S.P.J., M.O. and A.R.P.; analysis and interpretation of data: R.J.M., A.H.A., J.K., D.B., C.D., M.F.E., S.P.J., M.O., A.R.P. and M.E.; writing, review, and/or revision of the manuscript: R.J.M., A.H.A., J.K., D.B., C.D., M.F.E., S.P.J., M.O., A.R.P. and M.E.; administrative, technical, or material support: R.J.M., A.H.A., J.K., D.B., C.D., M.F.E., S.P.J., M.O., A.R.P. and M.E; study supervision: A.R.P. and M.E. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported in part through the Victorian State Government Operational Infrastructure Support, the National Health and Medical Research Council (NHMRC) of Australia project and development grants 1081373, 1092788 and 2014063. R.J.M is supported by a La Trobe University Postgraduate Research Scholarship (LTUPRS) and an Australian Government Research Training Program (RTP) Fees Offset Scholarship. M.E received funding from Ludwig Cancer Research and is a NHMRC Investigator grant recipient (1173814). A.R.P is an NHMRC Peter Doherty Early Career Fellow (1166447). M.F.E is a Victorian Cancer Agency Mid-Career Research Fellow (MCRF20018) and an AACR-Debbie’s Dream Foundation Gastric Cancer Award recipient (ID: 22-20-41-EISS).

Institutional Review Board Statement

All procedures and experiments were performed according to the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Approvals were granted from the La Trobe University Animal Ethics Committee (AEC15-37 approved 2015, AEC15-39 approved 2015, and AEC18069 approved 2018) and the Austin Health Animal Ethics Committee (A2016/05419 approved 2016 and A2017/05464 approved 2017).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author Matthias Ernst (matthias.ernst@onjcri.org.au).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Mantovani, A.; Marchesi, F.; Malesci, A.; Laghi, L.; Allavena, P. Tumour-associated macrophages as treatment targets in oncology. Nat. Rev. Clin. Oncol. 2017, 14, 399–416. [Google Scholar] [CrossRef] [PubMed]
  2. Poh, A.R.; Ernst, M. Targeting Macrophages in Cancer: From Bench to Bedside. Front. Oncol. 2018, 8, 49. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Dang, C.V. MYC on the Path to Cancer. Cell 2012, 149, 22–35. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Dang, C.V. c-Myc Target Genes Involved in Cell Growth, Apoptosis, and Metabolism. Mol. Cell Biol. 1999, 19, 1–11. [Google Scholar] [CrossRef] [Green Version]
  5. Rathmell, J.C. T Cell Myc-tabolism. Immunity 2011, 35, 845–846. [Google Scholar] [CrossRef] [Green Version]
  6. Dong, Y.; Tu, R.; Liu, H.; Qing, G. Regulation of cancer cell metabolism: Oncogenic MYC in the driver’s seat. Signal Transduct. Target. Ther. 2020, 5, 124. [Google Scholar] [CrossRef]
  7. Chen, H.; Liu, H.; Qing, G. Targeting oncogenic Myc as a strategy for cancer treatment. Signal Transduct. Target. Ther. 2018, 3, 5. [Google Scholar] [CrossRef] [Green Version]
  8. Felsher, D.W. MYC Inactivation Elicits Oncogene Addiction through Both Tumor Cell-Intrinsic and Host-Dependent Mechanisms. Genes Cancer 2010, 1, 597–604. [Google Scholar] [CrossRef]
  9. Schrier, P.I.; Peltenburg, L.T. Relationship between myc oncogene activation and MHC class I expression. Adv. Cancer Res. 1993, 60, 181–246. [Google Scholar]
  10. Dhanasekaran, R.; Deutzmann, A.; Mahauad-Fernandez, W.D.; Hansen, A.S.; Gouw, A.M.; Felsher, D.W. The MYC oncogene-the grand orchestrator of cancer growth and immune evasion. Nat. Rev. Clin. Oncol. 2021, 19, 23–36. [Google Scholar] [CrossRef]
  11. Han, H.; Jain, A.D.; Truica, M.I.; Izquierdo-Ferrer, J.; Anker, J.F.; Lysy, B.; Sagar, V.; Luan, Y.; Chalmers, Z.R.; Unno, K.; et al. Small-Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy. Cancer Cell 2019, 36, 483–497.e15. [Google Scholar] [CrossRef] [PubMed]
  12. Martinez, F.O.; Helming, L.; Milde, R.; Varin, A.; Melgert, B.N.; Draijer, C.; Thomas, B.; Fabbri, M.; Crawshaw, A.; Ho, L.P.; et al. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: Similarities and differences. Blood 2013, 121, e57–e69. [Google Scholar] [CrossRef] [PubMed]
  13. Pello, O.M.; De Pizzol, M.; Mirolo, M.; Soucek, L.; Zammataro, L.; Amabile, A.; Doni, A.; Nebuloni, M.; Swigart, L.B.; Evan, G.I.; et al. Role of c-MYC in alternative activation of human macrophages and tumor-associated macrophage biology. Blood 2012, 119, 411–421. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Pello, O.M.; Chèvre, R.; Laoui, D.; De Juan, A.; Lolo, F.; Andrés-Manzano, M.J.; Serrano, M.; Van Ginderachter, J.; Andrés, V. In Vivo Inhibition of c-MYC in Myeloid Cells Impairs Tumor-Associated Macrophage Maturation and Pro-Tumoral Activities. PLoS ONE 2012, 7, e45399. [Google Scholar] [CrossRef] [PubMed]
  15. Bae, S.; Park, P.S.U.; Lee, Y.; Mun, S.H.; Giannopoulou, E.; Fujii, T.; Lee, K.P.; Violante, S.N.; Cross, J.R.; Park-Min, K.-H. MYC-mediated early glycolysis negatively regulates proinflammatory responses by controlling IRF4 in inflammatory macrophages. Cell Rep. 2021, 35, 109264. [Google Scholar] [CrossRef]
  16. Clausen, B.E.; Burkhardt, C.; Reith, W.; Renkawitz, R.; Förster, I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999, 8, 265–277. [Google Scholar] [CrossRef]
  17. de Alboran, I.M.; O’Hagan, R.C.; Gärtner, F.; Malynn, B.; Davidson, L.; Rickert, R.; Rajewsky, K.; DePinho, R.A.; Alt, F.W. Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity 2001, 2001. 14, 45–55. [Google Scholar] [CrossRef] [Green Version]
  18. Tebbutt, N.C.; Giraud, A.S.; Inglese, M.; Jenkins, B.; Waring, P.; Clay, F.J.; Malki, S.; Alderman, B.M.; Grail, D.; Hollande, F.; et al. Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice. Nat. Med. 2002, 8, 1089–1097. [Google Scholar] [CrossRef]
  19. Thiem, S.; Eissmann, M.F.; Stuart, E.; Elzer, J.; Jonas, A.; Buchert, M.; Ernst, M. Inducible gene modification in the gastric epithelium of Tff1-CreERT2, Tff2-rtTA, Tff3-luc mice. Genesis 2016, 54, 626–635. [Google Scholar] [CrossRef]
  20. Flanagan, D.J.; Schwab, R.H.M.; Tran, B.M.; Phesse, T.J.; Vincan, E. Isolation and Culture of Adult Intestinal, Gastric, and Liver Organoids for Cre-recombinase-Mediated Gene Deletion. Methods Mol. Biol. 2019, 1576, 123–133. [Google Scholar]
  21. Eissmann, M.F.; Dijkstra, C.; Jarnicki, A.; Phesse, T.; Brunnberg, J.; Poh, A.R.; Etemadi, N.; Tsantikos, E.; Thiem, S.; Huntington, N.D.; et al. IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization. Nat. Commun. 2019, 10, 2735. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Allam, A.H.; Russell, S.M. Establishing a multiplex imaging panel to study T cell development in the thymus in mouse. STAR Protoc. 2022, 3, 101472. [Google Scholar] [CrossRef] [PubMed]
  23. Poh, A.R.; Love, C.G.; Chisanga, D.; Steer, J.H.; Baloyan, D.; Chopin, M.; Nutt, S.; Rautela, J.; Huntington, N.D.; Etemadi, N.; et al. Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy. Sci. Adv. 2022, 8, eabl7882. [Google Scholar] [CrossRef] [PubMed]
  24. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  25. Poh, A.R.; Love, C.G.; Masson, F.; Preaudet, A.; Tsui, C.; Whitehead, L.; Monard, S.; Khakham, Y.; Burstroem, L.; Lessene, G.; et al. Inhibition of Hematopoietic Cell Kinase Activity Suppresses Myeloid Cell-Mediated Colon Cancer Progression. Cancer Cell 2017, 31, 563–575.e5. [Google Scholar] [CrossRef] [Green Version]
  26. Poh, A.R.; Dwyer, A.R.; Eissmann, M.F.; Chand, A.L.; Baloyan, D.; Boon, L.; Murrey, M.W.; Whitehead, L.; O′Brien, M.; Lowell, C.A.; et al. Inhibition of the SRC Kinase HCK Impairs STAT3-Dependent Gastric Tumor Growth in Mice. Cancer Immunol. Res. 2020, 8, 428–435. [Google Scholar] [CrossRef] [Green Version]
  27. Abram, C.L.; Roberge, G.L.; Hu, Y.; Lowell, C.A. Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice. J. Immunol. Methods 2014, 408, 89–100. [Google Scholar] [CrossRef] [Green Version]
  28. Poh, A.R.; O’Donoghue, R.J.J.; Ernst, M.; Putoczki, T.L. Mouse models for gastric cancer: Matching models to biological questions. J. Gastroenterol. Hepatol. 2016, 31, 1257–1272. [Google Scholar] [CrossRef]
  29. Jenkins, B.J.; Grail, D.; Nheu, T.; Najdovska, M.; Wang, B.; Waring, P.; Inglese, M.; McLoughlin, R.M.; Jones, S.A.; Topley, N.; et al. Hyperactivation of Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-β signaling. Nat. Med. 2005, 11, 845–852. [Google Scholar] [CrossRef]
  30. Meškytė, E.M.; Keskas, S.; Ciribilli, Y. MYC as a Multifaceted Regulator of Tumor Microenvironment Leading to Metastasis. Int. J. Mol. Sci. 2020, 21, 7710. [Google Scholar] [CrossRef]
  31. Jiang, K.; Zhang, Q.; Fan, Y.; Li, J.; Zhang, J.; Wang, W.; Fan, J.; Guo, Y.; Liu, S.; Hao, D.; et al. MYC inhibition reprograms tumor immune microenvironment by recruiting T lymphocytes and activating the CD40/CD40L system in osteosarcoma. Cell Death Discov. 2022, 8, 117. [Google Scholar] [CrossRef] [PubMed]
  32. Casey, S.C.; Tong, L.; Li, Y.; Do, R.; Walz, S.; Fitzgerald, K.N.; Gouw, A.M.; Baylot, V.; Gütgemann, I.; Eilers, M.; et al. MYC regulates the antitumor immune response through CD47 and PD-L1. Science 2016, 352, 227–231. [Google Scholar] [CrossRef] [PubMed]
  33. Murray, P.J.; Allen, J.E.; Biswas, S.K.; Fisher, E.A.; Gilroy, D.W.; Goerdt, S.; Gordon, S.; Hamilton, J.A.; Ivashkiv, L.B.; Lawrence, T.; et al. Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines. Immunity 2014, 41, 14–20. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Mosser, D.M.; Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 2008, 8, 958–969. [Google Scholar] [CrossRef]
  35. Sica, A.; Larghi, P.; Mancino, A.; Rubino, L.; Porta, C.; Totaro, M.G.; Rimoldi, M.; Biswas, S.K.; Allavena, P.; Mantovani, A. Macrophage polarization in tumour progression. Semin. Cancer Biol. 2008, 18, 349–355. [Google Scholar] [CrossRef]
  36. Sica, A.; Mantovani, A. Macrophage plasticity and polarization: In vivo veritas. J. Clin. Investig. 2012, 122, 787–795. [Google Scholar] [CrossRef]
  37. Chawla, A. Control of macrophage activation and function by PPARs. Circ. Res. 2010, 106, 1559–1569. [Google Scholar] [CrossRef]
  38. Yu, T.; Gan, S.; Zhu, Q.; Dai, D.; Li, N.; Wang, H.; Chen, X.; Hou, D.; Wang, Y.; Pan, Q.; et al. Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24. Nat. Commun. 2019, 10, 4353. [Google Scholar] [CrossRef] [Green Version]
  39. Lawrence, T.; Natoli, G. Transcriptional regulation of macrophage polarization: Enabling diversity with identity. Nat. Rev. Immunol. 2011, 11, 750–761. [Google Scholar] [CrossRef]
  40. Rahman, S.M.; Janssen, R.C.; Choudhury, M.; Baquero, K.C.; Aikens, R.M.; de la Houssaye, B.A.; Friedman, J.E. CCAAT/enhancer-binding protein β (C/EBPβ) expression regulates dietary-induced inflammation in macrophages and adipose tissue in mice. J. Biol. Chem. 2012, 287, 34349–34360. [Google Scholar] [CrossRef] [Green Version]
  41. Chistiakov, D.A.; Myasoedova, V.A.; Revin, V.V.; Orekhov, A.N.; Bobryshev, Y.V. The impact of interferon-regulatory factors to macrophage differentiation and polarization into M1 and M2. Immunobiology 2018, 223, 101–111. [Google Scholar] [CrossRef] [PubMed]
  42. Liao, X.; Sharma, N.; Kapadia, F.; Zhou, G.; Lu, Y.; Hong, H.; Paruchuri, K.; Mahabeleshwar, G.H.; Dalmas, E.; Venteclef, N.; et al. Krüppel-like factor 4 regulates macrophage polarization. J. Clin. Investig. 2011, 121, 2736–2749. [Google Scholar] [CrossRef] [PubMed]
  43. Dorrington, M.G.; Fraser, I.D.C. NF-κB Signaling in Macrophages: Dynamics, Crosstalk, and Signal Integration. Front. Immunol. 2019, 10, 705. [Google Scholar] [CrossRef] [PubMed]
  44. Pyonteck, S.M.; Akkari, L.; Schuhmacher, A.J.; Bowman, R.L.; Sevenich, L.; Quail, D.F.; Olson, O.C.; Quick, M.L.; Huse, J.T.; Teijeiro, V.; et al. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat. Med. 2013, 19, 1264–1272. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Kaneda, M.M.; Cappello, P.; Nguyen, A.V.; Ralainirina, N.; Hardamon, C.R.; Foubert, P.; Schmid, M.C.; Sun, P.; Mose, E.; Bouvet, M.; et al. Macrophage PI3Kγ Drives Pancreatic Ductal Adenocarcinoma Progression. Cancer Discov. 2016, 6, 870–885. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Poh, A.R.; O’Brien, M.; Chisanga, D.; He, H.; Baloyan, D.; Traichel, J.; Dijkstra, C.; Chopin, M.; Nutt, S.; Whitehead, L.; et al. Inhibition of HCK in myeloid cells restricts pancreatic tumor growth and metastasis. Cell Rep. 2022, 41, 111479. [Google Scholar] [CrossRef]
  47. Pello, O.M. Macrophages and c-Myc cross paths. OncoImmunology 2016, 5, e1151991. [Google Scholar] [CrossRef] [Green Version]
  48. Cheng, S.-C.; Quintin, J.; Cramer, R.A.; Shepardson, K.M.; Saeed, S.; Kumar, V.; Giamarellos-Bourboulis, E.J.; Martens, J.H.A.; Rao, N.A.; Aghajanirefah, A.; et al. mTOR- and HIF-1α–mediated aerobic glycolysis as metabolic basis for trained immunity. Science 2014, 345, 1250684. [Google Scholar] [CrossRef] [Green Version]
  49. Doe, M.R.; Ascano, J.M.; Kaur, M.; Cole, M.D. Myc Posttranscriptionally Induces HIF1 Protein and Target Gene Expression in Normal and Cancer Cells. Cancer Res. 2012, 72, 949–957. [Google Scholar] [CrossRef] [Green Version]
  50. Wang, T.; Liu, H.; Lian, G.; Zhang, S.-Y.; Wang, X.; Jiang, C. HIF1α-Induced Glycolysis Metabolism Is Essential to the Activation of Inflammatory Macrophages. Mediat. Inflamm. 2017, 2017, 9029327. [Google Scholar] [CrossRef] [Green Version]
  51. Zhong, L.; Zhang, Y.; Li, M.; Song, Y.; Liu, D.; Yang, X.; Yang, D.; Qu, H.; Lai, L.; Wang, Q.; et al. E3 ligase FBXW7 restricts M2-like tumor-associated macrophage polarization by targeting c-Myc. Aging 2020, 12, 24394–24423. [Google Scholar] [CrossRef] [PubMed]
  52. Li, J.; Liang, Y.; Lv, H.; Meng, H.; Xiong, G.; Guan, X.; Chen, X.; Bai, Y.; Wang, K. miR-26a and miR-26b inhibit esophageal squamous cancer cell proliferation through suppression of c-MYC pathway. Gene 2017, 625, 1–9. [Google Scholar] [CrossRef]
  53. Sahu, S.K.; Kumar, M.; Chakraborty, S.; Banerjee, S.K.; Kumar, R.; Gupta, P.; Jana, K.; Gupta, U.D.; Ghosh, Z.; Kundu, M.; et al. MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection. PLoS Pathog. 2017, 13, e1006410. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Trumpp, A.; Refaeli, Y.; Oskarsson, T.; Gasser, S.; Murphy, M.; Martin, G.R.; Bishop, J.M. c-Myc regulates mammalian body size by controlling cell number but not cell size. Nature 2001, 414, 768–773. [Google Scholar] [CrossRef] [PubMed]
  55. Kortlever, R.M.; Sodir, N.; Wilson, C.; Burkhart, D.L.; Pellegrinet, L.; Swigart, L.B.; Littlewood, T.; Evan, G. Myc Cooperates with Ras by Programming Inflammation and Immune Suppression. Cell 2017, 171, 1301–1315.e14. [Google Scholar] [CrossRef]
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Figure 1. LysMCre-mediated reduction in Myc expression in mature macrophages. Expression of Myc in peritoneal, bone-marrow-derived, and splenic macrophages isolated from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl mice. n ≥ 3 mice per group. Data represent mean ± SEM; * p < 0.05, ** p < 0.01, with statistical significance determined by an unpaired Student’s t-test.
Figure 1. LysMCre-mediated reduction in Myc expression in mature macrophages. Expression of Myc in peritoneal, bone-marrow-derived, and splenic macrophages isolated from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl mice. n ≥ 3 mice per group. Data represent mean ± SEM; * p < 0.05, ** p < 0.01, with statistical significance determined by an unpaired Student’s t-test.
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Figure 2. Tumor growth remains refractory to Myc ablation in host macrophages. (A) Myc gene expression in gastric tumors of LysM+/+;Mycfl/fl;Gp130F/F and LysMCre/+;Mycfl/fl;Gp130F/F mice. n = 4 mice per group. (B) Mass of gastric tumors in LysM+/+;Mycfl/fl;Gp130F/F and LysMCre/+;Mycfl/fl;Gp130F/F mice collected at 100 days of age. Each symbol represents an individual mouse. n = 12 mice per group. (C) Myc gene expression in subcutaneous KPT gastric tumor organoids of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n = 4 mice per group. (D) Mass of subcutaneous KPT gastric tumor organoids from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Mice were collected at 3 weeks following tumor organoid injection. Each symbol represents an individual mouse. n ≥ 4 mice per group. (E) Myc gene expression in TAMs isolated from subcutaneous B16F10 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n ≥ 3 mice per group. (F) Mass of subcutaneous B16F10 tumors from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Mice were collected at 2 weeks following tumor cell injection. Each symbol represents an individual mouse. n = 11 mice per group. (G) Myc gene expression in TAMs isolated from subcutaneous MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n ≥ 3 mice per group. (H) Mass of subcutaneous MC38 tumors from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Mice were collected at 2 weeks following tumor cell injection. Each symbol represents an individual mouse. n ≥ 13 mice per group. Data represent mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, with statistical significance determined by an unpaired Student’s t-test.
Figure 2. Tumor growth remains refractory to Myc ablation in host macrophages. (A) Myc gene expression in gastric tumors of LysM+/+;Mycfl/fl;Gp130F/F and LysMCre/+;Mycfl/fl;Gp130F/F mice. n = 4 mice per group. (B) Mass of gastric tumors in LysM+/+;Mycfl/fl;Gp130F/F and LysMCre/+;Mycfl/fl;Gp130F/F mice collected at 100 days of age. Each symbol represents an individual mouse. n = 12 mice per group. (C) Myc gene expression in subcutaneous KPT gastric tumor organoids of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n = 4 mice per group. (D) Mass of subcutaneous KPT gastric tumor organoids from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Mice were collected at 3 weeks following tumor organoid injection. Each symbol represents an individual mouse. n ≥ 4 mice per group. (E) Myc gene expression in TAMs isolated from subcutaneous B16F10 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n ≥ 3 mice per group. (F) Mass of subcutaneous B16F10 tumors from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Mice were collected at 2 weeks following tumor cell injection. Each symbol represents an individual mouse. n = 11 mice per group. (G) Myc gene expression in TAMs isolated from subcutaneous MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n ≥ 3 mice per group. (H) Mass of subcutaneous MC38 tumors from LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Mice were collected at 2 weeks following tumor cell injection. Each symbol represents an individual mouse. n ≥ 13 mice per group. Data represent mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, with statistical significance determined by an unpaired Student’s t-test.
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Figure 3. Inhibition of Myc signaling in TAMs does not reduce tumor immune suppression. qPCR analysis on (A) Gp130F/F, (B) KPT, (C) B16F10 and (D) MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts for genes associated with immune suppression (i.e., Il4, Il10, Il13, Arg1, Ym1, Mrc1, Tgfβ) or immune activation (i.e., Il1β, Il12, Tnfα, Nos2, Ifnγ, GzmB, Prf1). n = 4 mice per group. Data represent mean ± SEM.
Figure 3. Inhibition of Myc signaling in TAMs does not reduce tumor immune suppression. qPCR analysis on (A) Gp130F/F, (B) KPT, (C) B16F10 and (D) MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts for genes associated with immune suppression (i.e., Il4, Il10, Il13, Arg1, Ym1, Mrc1, Tgfβ) or immune activation (i.e., Il1β, Il12, Tnfα, Nos2, Ifnγ, GzmB, Prf1). n = 4 mice per group. Data represent mean ± SEM.
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Figure 4. Genetic reduction of Myc in TAMs does not inhibit alternative macrophage polarization or stimulate adaptive anti-tumor immunity. (A,B) Quantification of TAMs and CD8+ T-cells in subcutaneous (A) B16F10 and (B) MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Each symbol represents an individual mouse. n ≥ 6 mice per group. (C,D) qPCR analysis on CD45+CD11b+F4/80+Ly6C−LygG− TAMs and CD45+TCRβ+CD8+ T-cells isolated from subcutaneous (C) B16F10 and (D) MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n ≥ 3 mice per group. Data represent mean ± SEM; * p < 0.05, ** p < 0.01, with statistical significance determined by an unpaired Student’s t-test.
Figure 4. Genetic reduction of Myc in TAMs does not inhibit alternative macrophage polarization or stimulate adaptive anti-tumor immunity. (A,B) Quantification of TAMs and CD8+ T-cells in subcutaneous (A) B16F10 and (B) MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. Each symbol represents an individual mouse. n ≥ 6 mice per group. (C,D) qPCR analysis on CD45+CD11b+F4/80+Ly6C−LygG− TAMs and CD45+TCRβ+CD8+ T-cells isolated from subcutaneous (C) B16F10 and (D) MC38 tumors of LysM+/+;Mycfl/fl and LysMCre/+;Mycfl/fl hosts. n ≥ 3 mice per group. Data represent mean ± SEM; * p < 0.05, ** p < 0.01, with statistical significance determined by an unpaired Student’s t-test.
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Morrow, R.J.; Allam, A.H.; Konecnik, J.; Baloyan, D.; Dijkstra, C.; Eissmann, M.F.; Jacob, S.P.; O’Brien, M.; Poh, A.R.; Ernst, M. Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages. Cells 2022, 11, 4104. https://doi.org/10.3390/cells11244104

AMA Style

Morrow RJ, Allam AH, Konecnik J, Baloyan D, Dijkstra C, Eissmann MF, Jacob SP, O’Brien M, Poh AR, Ernst M. Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages. Cells. 2022; 11(24):4104. https://doi.org/10.3390/cells11244104

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Morrow, Riley J., Amr H. Allam, Josh Konecnik, David Baloyan, Christine Dijkstra, Moritz F. Eissmann, Saumya P. Jacob, Megan O’Brien, Ashleigh R. Poh, and Matthias Ernst. 2022. "Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages" Cells 11, no. 24: 4104. https://doi.org/10.3390/cells11244104

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