Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages

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.


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 antibodydependent 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.

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 LysM Cre/+ strain [16] was crossed with the Myc fl/fl strain [17] to generate LysM Cre/+ ;Myc fl/fl and LysM +/+ ;Myc fl/fl mice [14]. Where indicated, LysM Cre/+ ;Myc fl/fl and LysM +/+ ;Myc fl/fl mice were additionally crossed with Gp130 F/F mice [18] to generate LysM Cre/+ ;Myc fl/fl ;Gp130 F/F and LysM +/+ ;Myc fl/fl ;Gp130 F/F animals. The Tff1 CreERT2 strain [19] was crossed with Myc fl/fl ; Gp130 F/F animals to generate Tff1 CreERT2 ;Myc fl/fl ;Gp130 F/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.
LysM +/+ ;Myc fl/fl ;Gp130 F/F and LysM Cre/+ ;Myc fl/fl ;Gp130 F/F animals were euthanized at 100 days of age. Stomachs were dissected longitudinally along the greater curvature, and gastric tumors were dissected and weighed.

Ablation of Gastric Epithelial Cells via Tamoxifen Treatment
Ninety-day-old Tff1 CreERT2 ;Myc fl/fl ;Gp130 F/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.

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% H 2 O 2 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 dH 2 O, 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. 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.

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 FACSisolated 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−∆∆CT method [24]. The Taqman probes used were mouse 18s (Mm04277571_s1), Gapdh (Mm99999915_g1),

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.

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.

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.

Quantification and Statistical Analysis
All experiments were performed at least twice with a minimum of three age-and sexmatched 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.

Myc Expression Is Reduced in Macrophages of LysM Cre/+ ;Myc fl/fl Mice
The transgenic LysM Cre/+ knock-in strain enables conditional depletion of Myc gene expression in macrophages of Myc fl/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 LysM Cre/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl mice. We observed an 80-90% reduction in Myc expression in these cells compared to cells purified from LysM Cre transgene-deficient LysM +/+ ;Myc fl/fl littermate controls (Figure 1). Consistent with previous observations of limited Cre recombinase activity conferred by LysM Cre in splenic myeloid cells [27], we confirmed a lack of quantitative Myc ablation in these cells (Figure 1).

Myc Expression Is Reduced in Macrophages of LysM Cre/+ ;Myc fl/fl Mice
The transgenic LysM Cre/+ knock-in strain enables conditional depletion of Myc gene expression in macrophages of Myc fl/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 LysM Cre/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl mice. We observed an 80-90% reduction in Myc expression in these cells compared to cells purified from LysM Cre transgene-deficient LysM +/+ ;Myc fl/fl littermate controls (Figure 1). Consistent with previous observations of limited Cre recombinase activity conferred by LysM Cre in splenic myeloid cells [27], we confirmed a lack of quantitative Myc ablation in these cells (Figure 1).

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 +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl mice to the Gp130 F/F mouse model. In Gp130 F/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 +/+ ;Myc fl/fl ;Gp130 F/F mice (Figure 2A and Supplementary Figure  S1A,B). However, we did not observe a difference in tumor burden between LysM +/+ ;Myc fl/fl ;Gp130 F/F and LysM Cre/+ ;Myc fl/fl ;Gp130 F/F littermates ( Figure 2B). By contrast, tamoxifeninduced Cre-mediated ablation of Myc in the gastric epithelium of tumor-bearing

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 +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl mice to the Gp130 F/F mouse model. In Gp130 F/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 +/+ ;Myc fl/fl ;Gp130 F/F mice (Figure 2A and Supplementary Figure S1A,B). However, we did not observe a difference in tumor burden between LysM +/+ ;Myc fl/fl ;Gp130 F/F and LysM Cre/+ ;Myc fl/fl ;Gp130 F/F littermates ( Figure 2B). By contrast, tamoxifen-induced Cre-mediated ablation of Myc in the gastric epithelium of tumorbearing Tff1 CreERT2 ;Myc fl/fl ;Gp130 F/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 +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl hosts using tumor organoids derived from invasive gastric adenocarcinomas of Kras G12D/+ ;Pik3ca H1047R/+ ;Trp53 R172H/+ (KPT) mice. We confirmed reduced Myc expression in KPT tumors excised from LysM Cre/+ ;Myc fl/fl hosts; however, these tumors were of a comparable size to those collected from Myc-proficient LysM +/+ ;Myc fl/fl hosts ( Figure 2C,D). reduction in Myc expression in LysM ;Myc TAMs, we could not detect differences i the growth of B16F10 tumors between LysM +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl hosts (Figur 2E,F). In contrast, reduced Myc expression in MC38 TAMs coincided with larger tumo in LysM Cre/+ ;Myc fl/fl hosts compared to their LysM +/+ ;Myc fl/fl littermates ( Figure 2G,H). Co lectively, our results suggest that Myc ablation in host macrophages does not attenua tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site develops, or the stage along the tumor progression cascade.  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 LysM Cre/+ ;Myc fl/fl TAMs, we could not detect differences in the growth of B16F10 tumors between LysM +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl hosts ( Figure 2E, F.). In contrast, reduced Myc expression in MC38 TAMs coincided with larger tumors in LysM Cre/+ ;Myc fl/fl hosts compared to their LysM +/+ ;Myc fl/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.

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 Gp130 F/F , KPT, B16F10, or MC38 models that arose in either LysM +/+ ;Myc fl/fl or LysM Cre/+ ;Myc fl/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 +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl hosts. We observed a comparable abundance of TAMs and CD8 + T-cells in B16F10 tumors between LysM +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl hosts ( Figure 4A). In line with the increased tumor burden observed with MC38 tumor cells in LysM Cre/+ ;Myc fl/fl hosts, we also observed an increased proportion of TAMs in these tumors compared to LysM +/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl hosts to determine whether their endotype would differ from TAMs associated with tumors from LysM +/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl and LysM +/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl and LysM +/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl hosts.

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.
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 LysM Cre/+ ;Myc fl/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 LysM Cre/+ ;Myc fl/fl hosts. Importantly, we also failed to document increased anti-tumor activity despite less than 20% residual Myc expression in TAMs or tumors of LysM Cre/+ ;Myc fl/fl hosts in the MC38 and KPT allograft models, as well as in the endogenous Gp130 F/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 LysM Cre/+ transgene (Lyz2 tm1(cre)Ifo ) [16] and Myc fl/fl allele (Myc tm2Fwa ) [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 +/+ ;Myc fl/fl and LysM Cre/+ ;Myc fl/fl hosts with either altered abundance or activity of cytotoxic T-cells, suggesting that the increased tumor burden in LysM Cre/+ ;Myc fl/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 Tff1 CreERT2 ;Myc fl/fl ;Gp130 F/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.