Myeloid Mir34a suppresses tumor formation and progression in Apc
Min/+ mice
To determine whether myeloid Mir34a affects intestinal tumor formation, we crossed LysM-Cre/Mir34afl/fl mice with adenoma-prone ApcMin/+ mice on a C57BL/6 background to generate ApcMin/+ mice with deletion of Mir34a specifically in myeloid cells (ApcMin/+; Mir34aΔMye). At the age of 18 weeks, these mice displayed a more than 2-fold increase in the total number of intestinal neoplasms when compared to ApcMin/+ control mice (Fig. 1A–C). This effect was not observed in the colon (Fig. S1A–C). In addition, the number of high-grade adenomas was increased in age-matched ApcMin/+; Mir34aΔMye mice when compared to control mice (Fig. 1D–G). While neoplasms in Mir34a-proficient ApcMin/+ mice were predominantly low-grade adenomas and not invasive, in myeloid Mir34a-deficient mice, approximately 52% of neoplasms progressed to high-grade adenomas and 11% developed into invasive carcinomas (Fig. 1E–G). We also observed that, in age-matched, 18-week-old ApcMin/+; Mir34aΔMye mice, the total length of the small intestine, as well as the width and depth of the crypts, were significantly increased (Fig. S1D–F). In contrast, the total length and architecture of crypts in the colon were similar in both genotypes (Fig. S1G–I). However, as the increase in length was in the range of 9%, it could not account for the increased number of neoplasms observed in the myeloid Mir34a-deficient mice. Taken together, Mir34a-deficiency in the myeloid lineage enhanced tumor formation and progression in the small intestine of ApcMin/+ mice, suggesting that a physiological function of myeloid Mir34a is to suppress intestinal tumor initiation and progression.
Fig. 1: Myeloid Mir34a suppresses tumor formation and progression in ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Representative macroscopic images of polyps in resected small intestines of 18-week-old ApcMin/+ mice with the indicated genotypes. Scale bar: 2 cm. Representative H&E-stained “swiss-roll” sections of the small intestine of 18-week-old ApcMin/+ mice. Scale bar: 800 µm. B Quantification of the number of neoplasms within intestinal regions of 18-week-old ApcMin/+ mice with the indicated genotypes (n = 9 per genotype). C Sum of neoplasia area stratified by small intestinal region per mouse of ApcMin/+ mice with the indicated genotypes (n = 9 per genotype). D Representative pictures of different tumor stages in 18-week-old ApcMin/+ mice with the indicated genotypes. Scale bar: 200 µm. Red arrows correspond to invasive adenocarcinomas. E–G Quantification of different tumor stages and tumor number in neoplasms from the small intestine in 18-week-old ApcMin/+ mice with the indicated genotypes (n = 4 per genotype). SI small intestine. B, C, F Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.
Myeloid Mir34a modulates intestinal TME in Apc
Min/+ mice
Next, we determined whether loss of myeloid Mir34a affects cellular properties within intestinal tumors and the distribution of cell types in the TME of ApcMin/+ mice. Indeed, an increase of Ki-67-positive cells and a lower number of cleaved-caspase 3-positive tumor cells in intestinal and colonic neoplasms was observed in 18-week-old ApcMin/+ mice with myeloid Mir34a-deficiency when compared to their ApcMin/+ control littermates (Figs. 2A and S2A). Therefore, loss of myeloid Mir34a increases proliferation and decreases apoptosis of tumor cells. Furthermore, a significant increase in MPO+ neutrophils and F4/80+ macrophages in tumors and in their vicinity was detected in myeloid Mir34a-deficient ApcMin/+ mice (Fig. 2A). Therefore, myeloid Mir34a may suppress the accumulation of macrophages and neutrophils in the TME of intestinal tumors.
Fig. 2: Myeloid Mir34a modulates intestinal TME in ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Immunohistochemical detection of Ki-67, cleaved-caspase 3, F4/80 and Myeloperoxidase (MPO) in neoplasms from the small intestine in 18-weeks-old ApcMin/+ mice with the indicated genotypes (left panel). Scale bars: 40 µm. Quantification of Ki-67+, cleaved-caspase 3+, F4/80+ and MPO+ cells (right panel). B QPCR detection of the indicated mRNAs in neoplasms from the small intestine in 18-week-old ApcMin/+ mice with the indicated genotypes. A, B Three mice per genotype were evaluated. Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ***p < 0.001.
Next, we investigated whether myeloid Mir34a might influence the expression of molecules involved in the recruitment and polarization of myeloid cells. Indeed, intestinal neoplasms showed an up-regulation of mRNAs encoding myeloid cell recruitment-related cytokines (Ccr2, Ccl2, Cxcl10 and Cxcr3), as well as neutrophil recruitment-associated cytokines (Cxcr2, Cxcl1 and Cxcl2) in 18-week-old ApcMin/+; Mir34aΔMye mice when compared to control mice (Fig. 2B). These findings indicate that cytokines, which exert myeloid cell-recruitment functions, are secreted to a higher extent in neoplasms with Mir34a-deficient myeloid cells.
Additionally, the expression of genes associated with tumor-promoting M2-like TAMs, such as Arg1, Mrc1 and Ym-1 mRNAs, was elevated in neoplasms from ApcMin/+; Mir34aΔMye mice, whereas the expression of the marker Nos2, characteristic for anti-tumorigenic M1-like TAMs, was reduced when compared with neoplasms from age-matched APCMin/+ ; Mir34afl/fl mice (Fig. 2B). These results imply that myeloid Mir34a maintains intra-tumoral TAMs in an M1-like state through regulating the expression of polarization-related cytokines.
Furthermore, we detected an increased expression of several known Mir34a target mRNAs, such as Csf1r, Pd-l1, Mmp9, Ccl22, and c-Myc, in neoplasms of ApcMin/+; Mir34aΔMye mice (Fig. 2B). The concomitant increase of these Mir34a targets, including invasion-related factors, such as Mmp9 and c-Myc, the macrophage polarization-related factor, Csf1r, and immune-regulatory factors, such as Pd-l1, and Ccl22, might play an important role in driving the enhanced invasiveness of adenomas observed in ApcMin/+; Mir34aΔMye mice. Taken together, these findings suggest that myeloid Mir34a is critical for maintaining a tumor-suppressive state of myeloid cells within the TME of ApcMin/+ mice.
Mir34a affects myeloid polarization and T-cell recruitment in 18-week-old Apc
Min/+ mice
Arginase1 (Arg1) and nitric oxide synthase 2 (Nos2) are known markers for pro-tumorigenic, M2/N2-like (Arg1) and anti-tumorigenic, M1/N1-like (Nos2) polarization states of TAMs and TANs [24,25,26]. In order to determine the polarization state of TAMs and TANs, we next utilized indirect immunofluorescence detection to evaluate the frequency of Arg1+ and Nos2+ myeloid cells within neoplasms. Co-immunostaining of intestinal neoplasms revealed a significant increase by approximately 20% in the number of F4/80+Arg1+ cells and a significant decrease by about 13% in the number of F4/80+Nos2+ cells in age-matched ApcMin/+; Mir34aΔMye mice (Figs. 3A and S3A, B). Similar results were obtained by immunohistochemical detection of single markers (Fig. S2B). Surprisingly, we observed an increase in Nos2-positive MPO+ neutrophils in the tumor tissue of myeloid Mir34a-deficient mice, whereas Arg1-positive MPO+ cells were decreased (Figs. 3B and S3C, D).
Fig. 3: Mir34a affects myeloid polarization and T-cell recruitment in 18-week-old ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Representative pictures of macrophages in the small intestine of 18-week-old ApcMin/+ mice with the indicated genotypes. F4/80 (red), Arg1 (green), Nos2 (green), DAPI (blue), and dp (double positive, yellow). Scale bar: 20 µm. Quantification of F4/80+Arg1+ and F4/80+Nos2+ cells. B Representative pictures of neutrophils in the small intestine of 18-week-old ApcMin/+ mice with the indicated genotypes. MPO (red), Arg1 (green), Nos2 (green), DAPI (blue), and dp (double positive, yellow). Scale bar: 20 µm. Quantification of MPO+Arg1+ and MPO+Nos2+ cells. C Representative pictures of Granzyme-B-positive cytotoxic T-cells and Foxp3-positive regulatory T-cells in the small intestine of 18-week-old ApcMin/+ mice with the indicated genotypes. Granzyme-B (red), CD8 (green), Foxp3 (red), CD4 (green), DAPI (blue), and dp (double positive, yellow). Scale bar: 20 µm. Quantification of CD8+GzmB+ and CD4+Foxp3+ cells. A–C Three mice per genotype were evaluated. Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Next, we investigated whether the increase in M2-like TAMs due to myeloid Mir34a-deficiency affects T-cell infiltration and composition in 18-week-old ApcMin/+ mice. Quantification of CD3+CD8+ T-cells showed a significant decrease in 18-week-old ApcMin/+; Mir34aΔMye mice when compared to control mice (Fig. S4A). On the contrary, we observed an increase of CD3+CD4+ T-cells in the tumors of myeloid Mir34a-deficient mice (Fig. S4B). To further characterize the functional state of these T-cell populations, we next evaluated tumors for cytotoxic CD8+ T-cells and regulatory T-cells (Tregs). An increase of CD4+Foxp3+T-cells ( = Tregs) was observed in adenomas of myeloid Mir34a-deficient mice, whereas Granzyme-B-positive CD8+ T-cells were significantly decreased (Figs. 3C and S4C, D). Therefore, myeloid Mir34a-deficiency skews the T-cell compartment towards an immunosuppressive, pro-tumorigenic state.
Overall, myeloid-specific loss of Mir34a leads to a shift towards anti-inflammatory, tumor-supportive M2-like macrophages and immune-suppressive CD4+Foxp3+ Treg cells, while simultaneously driving the polarization of pro-inflammatory N1-like neutrophils. Therefore, myeloid Mir34a suppresses intestinal tumor development of ApcMin/+ mice by maintaining the polarization of TAMs and TANs in an anti-tumorigenic state, which also affects tumor-associated T-cells, such as Tregs.
Mir34a regulates polarization in BMDMs
In order to analyze the function of myeloid Mir34a on the cellular level, we next isolated BMDMs from ApcMin/+; Mir34afl/fl and ApcMin/+; Mir34aΔMye mice. The deletion of Mir34a was confirmed on the level of pri-Mir34a and mature Mir34a expression in BMDMs (Fig. S5A). Mir34a-deficient BMDMs displayed an up-regulation of myeloid-specific recruitment and differentiation markers, which confirms the observations made in mice deficient for myeloid Mir34a (Fig. 4A). These results indicate that myeloid Mir34a regulates myeloid recruitment and differentiation. Also, a significant increase of tumor-promoting/myeloid cell polarization related genes, Arg1, Ym-1 and Mrc1, as well as a significant decrease of the tumor-restricting/myeloid cell polarization related Nos2 mRNA was observed, when compared to myeloid Mir34a-proficient BMDMs (Fig. 4A). In addition, up-regulation of M2-like macrophage-related mRNAs, such as Il-4 and Il-10, as well as several known Mir34a targets (Csf1r, Pd-l1, Mmp9, Ccl22, and c-Myc), was detected in Mir34a-deficient BMDMs, when compared to myeloid Mir34a-proficient BMDMs (Fig. 4A).
Fig. 4: Mir34a regulates polarization in BMDMs.The alternative text for this image may have been generated using AI.
A QPCR detection of the expression of the indicated Mir34a target mRNAs in BMDMs of ApcMin/+ mice with the indicated genotypes. B Expression of polarization-associated genes in BMDMs after IL-4 treatment for 4 h in ApcMin/+ mice with the indicated genotypes. C Expression of polarization-associated genes in BMDMs after LPS + IFN-γ treatment for 4 h in ApcMin/+ mice with the indicated genotypes. D Migration of BMDMs derived from the indicated ApcMin/+ mice after 4 and 24 h. E Percentage of wound closure after 24 h co-culture of BMDMs and CT26 cells. CM, conditioned media. A–E Three mice per genotype were evaluated. Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.
Next, we polarized BMDMs to an M1-like state by exposure to LPS and IFN-γ, as well as to an M2-like state by IL-4 treatment. IL-4 led to elevated levels of Arg1, Mrc1, and Ym-1 in Mir34a-deficient BMDMs when compared to Mir34a-proficient BMDMs (Figs. 4B and S5B–D). Therefore, myeloid Mir34a-deficiency augments pro-tumoral M2-like polarization in BMDMs, which is consistent with the in vivo effects of myeloid Mir34a-deficiency observed above. After treatment with LPS and IFN-γ, Mir34a-deficient BMDMs maintained elevated expression of Arg1 and Ym-1, while Nos2 showed a non-significant increase compared with Mir34a-proficient BMDMs (Figs. 4C and S5E). These observations imply that Mir34a functions to maintain an M1-like polarization and thereby suppresses tumor progression.
Furthermore, a modified Boyden chamber assay showed that BMDMs with Mir34a-deficiency harbor enhanced migration capabilities when compared to control BMDMs (Figs. 4D and S5F). In order to determine whether myeloid Mir34a may affect tumor cell migration, we co-cultured murine CT-26 CRC cells with Mir34a-proficient or Mir34a-deficient BMDMs isolated from ApcMin/+ mice (Figs. 4E and S5G). CT-26 cells co-cultured with myeloid Mir34a-deficient BMDMs closed a scratch wound more efficiently than the controls, implying that myeloid Mir34a may prevent CRC migration by regulating the secretion of factors that influence the migration of neighboring CRC cells. Taken together, the findings obtained by analyzing Mir34a-deficient BMDMs further support the conclusion, that myeloid Mir34a suppresses tumor progression by restricting the shift of TAMs towards a tumor-promoting M2-like phenotype, which enhances migration and presumably other tumorigenic features of CRC cells, such as invasion.
Myeloid Mir34a deletion decreases the survival time of Apc
Min/+ mice
Next, we determined whether myeloid Mir34a also influences the overall survival of ApcMin/+ mice. Indeed, ApcMin/+; Mir34aΔMye mice showed a shortened survival time with a median of 130 days, compared to 162 days in ApcMin/+ control mice (Fig. 5A). A similar shorter overall survival was observed in both male and female mice with myeloid Mir34a-deficiency (Fig. S6A), indicating that these effects of myeloid Mir34a do not depend on gender. In the moribund mice deletion of Mir34a in the myeloid lineage resulted in a 2-fold increase of the total number of neoplasms (Fig. 5B, C). Moreover, an increased frequency of tumors was found in all regions of the small intestine in ApcMin/+; Mir34aΔMye mice (Fig. 5D). Furthermore, the invasive carcinomas were only detected in the small intestine of ApcMin/+; Mir34aΔMye mice when compared to ApcMin/+; Mir34afl/fl mice (Figs. 5E–G and S6B). Therefore, myeloid Mir34a presumably suppresses progression of adenomas to invasive carcinomas. These differences only reached significance in the small intestine but not in the colon (Fig. S6C–E). In addition, an increase in Ki-67-positive cells and a decrease in apoptotic cells was detected in neoplasms from ApcMin/+; Mir34aΔMye mice (Figs. 5H, I and S6F), which might be responsible for the increase in the number and size of neoplasms. Altogether, these findings demonstrate that myeloid Mir34a inhibits tumor growth, delays adenoma progression and thereby presumably enhances overall survival in ApcMin/+ mice.
Fig. 5: Myeloid Mir34a deletion decreases survival time of ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Kaplan-Meier survival analysis of ApcMin/+ mice with the indicated genotypes. Results were compared with a log-rank test. B Representative macroscopic images of polyps in resected small intestines of moribund ApcMin/+ mice with the indicated genotypes (upper panel, Scale bar: 1 cm). Representative H&E stained “swiss-roll” sections of the small intestine of moribund ApcMin/+ mice (lower panel, Scale bar: 800 µm). C Quantification of the number of neoplasms stratified by small intestinal region per mouse of moribund ApcMin/+ mice with the indicated genotypes (n = 10 per genotype). D Sum of neoplasia area stratified by small intestinal region per mouse of ApcMin/+ mice with the indicated genotypes (n = 10 per genotype). Quantification of E tumor stage and F, G tumor number from the small intestine in moribund ApcMin/+ mice with the indicated genotypes (n = 5 per genotype). SI small intestine. Quantification of immunohistochemistry of H Ki-67 and I cleaved-caspase 3 in neoplasms from the small intestine in moribund ApcMin/+ mice with the indicated genotypes (n = 3 per genotype). C, D, F, H, I Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.
Myeloid Mir34a suppresses early adenoma formation in 5-week-old Apc
Min/+ mice
To investigate the effect of myeloid Mir34a on early-stage tumorigenesis in ApcMin/+ mice, we examined the number of aberrant crypt foci (ACF), which are considered to represent the initial stage of intestinal tumor development [27]: 5-week-old ApcMin/+; Mir34aΔMye mice exhibited a significantly elevated number of ACFs in both small intestine and colon (Figs. 6A, B and S7A). Moreover, we quantified early neoplastic transformation in ApcMin/+ mice using β-catenin and H&E staining: myeloid Mir34a-deficient ApcMin/+ mice showed an elevated rate of dysplasia initiation in SI when compared with control ApcMin/+ mice (Figs. 6C, D and S7B, C). Furthermore, the microadenoma area was increased in the small intestine of 5-week-old ApcMin/+; Mir34aΔMye mice when compared to their control littermates (Fig. 6E) presumably due to an increase in proliferation and a decrease in apoptosis (Fig. 6F–H). Additionally, the number and area of colonic micro-adenomas was also increased in 5-week-old ApcMin/+; Mir34aΔMye mice (Fig. 6I–K). In summary, our results indicate that myeloid Mir34a suppresses tumor initiation in the small intestine and colon of ApcMin/+ mice.
Fig. 6: Effect of myeloid Mir34a on early adenoma formation in 5-week-old ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Quantification of the number of ACFs per segment of small intestine in 5-week-old ApcMin/+ mice with the indicated genotypes (n = 4 per genotype). SI small intestine, ACF aberrant crypt foci. B Quantification of the number of ACFs in total colon in 5-week-old ApcMin/+ mice with the indicated genotypes (n = 4 per genotype). C Representative H&E stainings of micro-adenoma in small intestine in 5-week-old ApcMin/+ mice with the indicated genotypes. Scale bar: 100 µm. D Quantification of the number of micro-adenomas in the small intestine per mouse of 5-week-old ApcMin/+ mice with the indicated genotypes (n = 7 per genotype). E Sum of micro-adenoma area per mouse in the small intestine of 5-week-old ApcMin/+ mice with the indicated genotypes (n = 7 per genotype). F Representative pictures of Ki-67- and cleaved-caspase 3-positive stained cells in micro-adenomas from the small intestine of 5-week-old ApcMin/+ mice with the indicated genotypes. Scale bars: 50 µm. Quantification of G Ki-67 and H cleaved-caspase 3 in micro-adenomas from the small intestine in 5-week-old ApcMin/+ mice with the indicated genotypes (n = 3 per genotype). I Representative H&E staining pictures of micro-adenoma in colon of 5-week-old ApcMin/+ mice with the indicated genotypes. Scale bar: 50 µm. J Quantification of the number of micro-adenomas in colon per mouse of 5-week-old ApcMin/+ mice with the indicated genotypes (n = 8 per genotype). K Sum of micro-adenoma area per mouse in colon of 5-week-old ApcMin/+ mice with the indicated genotypes (n = 8 per genotype). A, B, D, E, G, H, J, K Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ns, not significant.
Myeloid Mir34a suppresses colon cancers in DSS-treated Apc
Min/+ mice
Next, we investigated the effect of myeloid Mir34a loss on colorectal cancer formation in ApcMin/+ mice, which can be induced by treatment with dextran sodium sulfate (DSS) [28]. We evaluated the colon-carcinoma burden of mice following treatment with 2% DSS for a 7-day cycle (Fig. 7A). ApcMin/+; Mir34aΔMye mice demonstrated a significant increase in both, number and size of colon cancers, as well as a rise in the proportion of Ki-67-positive cells (Figs. 7B–F and S8A). A concomitant decrease of cleaved-caspase 3-positive cells was observed, when compared to control mice. These findings indicate that the enhanced number and size of colon cancers in myeloid Mir34a-deficient ApcMin/+ mice is presumably driven by higher proliferation rates and less apoptosis. Additionally, mice with myeloid Mir34a-deficiency showed an increased number of high-grade adenomas and invasive adenocarcinomas, whereas the number of low-grade adenomas was decreased when compared to control littermates. Notably, invasive adenocarcinomas were only observed in myeloid Mir34a-deficient mice, indicative of a suppressive effect of myeloid Mir34a on cancer cell invasiveness (Figs. 7G–I and S8B).
Fig. 7: Myeloid Mir34a suppresses colon cancers in DSS-treated ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Schematic illustration of the experimental design. B Representative H&E stained “Swiss-roll” sections of 2% DSS treated ApcMin/+ mice. Scale bar: 2 mm. C Quantification of the number of neoplasms in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes (n = 6 per genotype). D Sum of neoplasia area in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes (n = 6 per genotype). Quantification of E Ki-67+ and F cleaved-caspase 3+ cells in neoplasms in 2% DSS treated ApcMin/+ mice with the indicated genotypes (n = 3 per genotype). G Representative pictures of different tumor stages in 2% DSS treated ApcMin/+ mice with the indicated genotypes. Scale bar: 100 µm. Red arrows correspond to invasive adenocarcinomas. H, I Quantification of different tumor stages and tumor number in neoplasms from colon in 2% DSS treated ApcMin/+ mice with the indicated genotypes (n = 6 per genotype). Quantification of J F4/80+ and K MPO+ cells in neoplasms from colon in 2% DSS treated ApcMin/+ mice with the indicated genotypes (n = 3 per genotype). C–F, H, J, K Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, not significant.
Collectively, these results showed that myeloid Mir34a-deficient mice are more susceptible to the formation of DSS-induced colon cancer, indicating a protective role of myeloid Mir34a against DSS-induced colon carcinomas in ApcMin/+ mice.
Mir34a influences myeloid polarization and T-cell recruitment in DSS-treated Apc
Min/+ mice
In order to determine whether myeloid Mir34a-deficiency affects the distribution of tumor-associated myeloid cells in DSS-induced colon cancers of ApcMin/+ mice, we next evaluated the number of F4/80- and MPO-positive cells. Interestingly, a different pattern than in 18-week-old mice without DSS-treatment was observed after treatment with 2% DSS, with higher numbers of F4/80+ macrophages, but a decrease in MPO+ neutrophiles in colon cancers from ApcMin/+; Mir34aΔMye mice when compared to ApcMin/+ mice, indicating that macrophages might play a more important role than neutrophils in colon cancer progression than in the formation of adenomas in the small intestine (Figs. 7J, K and S8C). Nevertheless, the number of Arg1+ cells was elevated, while the number of Nos2+ cells was reduced in colon cancers of Mir34a-deficient mice (Fig. S8C), which was also seen in the SI of 18-week-old mice (Fig. S2B). In support of a tumor suppressive effect of myeloid Mir34a in macrophages, we verified that there were significantly more F4/80+Arg1+ cells and fewer F4/80+Nos2+ cells in colon cancers of ApcMin/+; Mir34aΔMye mice (Figs. 8A, B and S9A, B). A similar pattern was observed in MPO+ neutrophils, with significantly more MPO+Arg1+ cells and fewer MPO+Nos2+ cells (Figs. 8C, D and S9C, D). To further determine whether myeloid Mir34a indeed affects T-cell populations and their functional state in DSS-induced colon cancers of ApcMin/+ mice, we evaluated the same markers as for the age-matched ApcMin/+ mice. We detected increased numbers of CD4+ T-cells and Foxp3+ Tregs in colon cancers from ApcMin/+; Mir34aΔMye mice when compared to ApcMin/+ control mice, while CD8+ T-cells and Granzyme-B+ cytotoxic T-cells were reduced (Figs. 8E–H and S10A–D).
Fig. 8: Mir34a influences myeloid polarization and T-cell recruitment in DSS-treated ApcMin/+ mice.The alternative text for this image may have been generated using AI.
A Quantification of F4/80+Arg1+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. B Quantification of F4/80+Nos2+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. C Quantification of MPO+Arg1+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. D Quantification of MPO+Nos2+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. E Quantification of CD3+CD8+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. F Quantification of CD3+CD4+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. G Quantification of CD8+GzmB+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. H Quantification of CD4+Foxp3+ cells in the colon of 2% DSS treated ApcMin/+ mice with the indicated genotypes. I Graphical summary of results and conclusions obtained in this study. A–H Three mice per genotype were evaluated. Mean values ± SEM are provided. Student’s t test was used to determine significance. *p < 0.05, **p < 0.01.
Therefore, loss of myeloid Mir34a function shifts both TAMs and TANs to pro-tumorigenic phenotypes in an inflammatory microenvironment. This shift in turn leads to a more immunosuppressive environment, presumably by increasing the number of CD4+Foxp3+ Treg cells. Overall, these findings show that myeloid Mir34a has a pivotal role in inhibiting macrophage infiltration into colon cancers and in suppressing pro-tumoral myeloid cell polarization in the TME of colon cancers induced by DSS in ApcMin/+ mice.
