Spleen mediates a distinct hematopoietic progenitor response supporting tumor-promoting myelopoiesis (original) (raw)
Myeloid-biased splenic early HSPCs in tumor-bearing mice. Hepatocellular carcinoma (HCC) represents a type of cancer that is associated with negligible or low levels of systemic myeloid differentiation cytokines but evident extramedullary HSPC activity (16). To investigate the cancer-associated EMH in such tumor settings, we used an orthotopic Hepa1-6 hepatoma mouse model (hereafter referred to as Hepa mice), which reproduces many features of human HCC (23, 24). In this model, the spleen was the major site of EMH (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI97973DS1). With slight splenomegaly, the spleens of Hepa mice accumulated both Linlo/–Sca-1+c-Kithi (LSK) and Linlo/–Sca-1–c-Kithi (LK) HSPCs in a progression-dependent manner (Supplemental Figure 1, C–E) and at levels comparable to those detected in other transplanted or genetic mouse tumor models (Supplemental Figure 1, F and G).
To describe and compare the cancer-associated hematopoietic alterations in BM and spleen, we initially performed competitive reconstitution experiments. BM from Hepa mice reconstituted myeloid cells and lymphocytes at levels similar to those of its control counterpart (Figure 1A). In contrast, the transfer of splenocytes from Hepa mice led to a rapid and more robust reconstitution compared with that seen in control spleen, with a myelopoiesis preference (Figure 1A). The fold changes of myeloid reconstitution peaked 2 weeks after transplantation, showing 6-fold greater chimerisms compared with control spleen and gradually decreased afterwards. In accordance with the boost of myeloid cell reconstitution, CFU assays showed a 15-fold increase in CFU-GM in the spleens of Hepa mice (Figure 1B), whereas the enrichment of multi-lineage CFU-granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) was insignificant. These findings suggested that the spleens of Hepa mice accommodated a myeloid-biased hematopoiesis facilitating rapid myeloid immune cell generation.
The myeloid-biased differentiation of splenic early HSPCs from Hepa mice. (A) Analysis of peripheral blood myeloid cell (CD11b+Gr-1+) and lymphocyte (CD3+ T and B220+ B cells) reconstitution chimerisms in recipient mice (BM, n = 7 mice per group; spleen, n = 9 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (B) Quantification of CFU-C activity in BM and spleens from control (n = 7) and Hepa (n = 8) mice. ***P < 0.001, by Student’s t test. BFU-E, burst-forming unit–erythroid. (C) CFU-S12 activity of 500 BM-derived (n = 15 recipients) or splenic (n = 21 recipients) LSK cells from Hepa mice. ***P < 0.001, by Student’s t test. (D) Scheme of the 2-step single-cell colony-forming assay, representative colonies, and the percentage of different types of lineage readouts of the secondary CFU-C. Scale bars: 500 μm. Numbers above the columns represent the sample size of the initiating single cells in each group. Results are shown as the mean ± SEM of mice in each group (A–C). Data are from 2 experiments (A and B) or 3 experiments, with cells pooled from 6 to 10 mice (C and D).
Heightened splenic myelopoiesis in cancer has been linked to the accumulation of multiple HSPC populations (13, 20, 21), but the functional preference of early HSPCs, the LSK cells, in the spleen of the tumor-bearing host remains unclear. These LSK cells are highly heterogeneous and contain various HSC and HPC subpopulations with different lineage potential (25–27). Although BM and splenic LSK cells from Hepa mice differentiated into FcγRloCD34+ common myeloid progenitors (CMPs) and FcγRhiCD34+ GMPs at similar kinetics in vitro (Supplemental Figure 1, H and I), LSK cells from the spleen produced markedly fewer trilineage splenic CFU in vivo (CFU-S12) (Figure 1C), suggesting a reduced proportion of multipotent HSCs (28). To corroborate the differentiation potential of splenic LSK cells at the single-cell level, we adopted a 2-step colony-forming assay (29). The daughter cells from more than 85% single BM LSK cells simultaneously formed both GM-type and megakaryocyte-erythrocyte–type (MegE-type) colonies, indicating that most BM LSK cells were multipotent (Figure 1D). In stark contrast, the daughter cells from more than 70% single splenic LSK cells formed GM-type colonies only, suggesting that the majority of splenic LSK cells were myeloid immune cell restricted. These findings indicate that the heightened splenic myelopoiesis in tumor-bearing mice is not only characterized by the accumulation of HSPCs, but is also associated with a significant myeloid skew within the LSK cell population.
Accumulation of GM-CSF–expressing LSK cells in the spleens of tumor-bearing mice. Emerging evidence has suggested that the biased lineage potential of LSK cells may be associated with their altered cytokine production capacity (30). Along this line, we found that a significantly higher proportion of splenic LSK cells from Hepa mice expressed GM-CSF, an important myeloid differentiation cytokine (Figure 2A). The enhanced GM-CSF expression was associated with upregulation of NF-κB activation and downregulation of p38 MAPK activation (Figure 2B; see complete unedited blots in the supplemental material). These GM-CSF–expressing HSPCs were commonly present in the spleens of tumor-bearing mice, including in another hepatoma model induced by _N_-nitrosodiethylamine (DEN) and CCl4, a transplantable melanoma model, and an intestinal neoplasia model caused by the Apc gene mutation (Supplemental Figure 2A). However, these cells were rarely detected in the BM, in the control spleen (Figure 2A), or in an EMH model induced by repeated bleeding (Supplemental Figure 2A). Moreover, we found that the level of GM-CSF was not increased in the serum or in the splenic microenvironment of Hepa mice (Supplemental Figure 2, B–E) and that another important cytokine, G-CSF, was also not increased in this setting (Supplemental Figure 2F). These findings suggested that the endogenous GM-CSF signal might be functionally significant for splenic LSK HSPC differentiation.
Accumulation of GM-CSF–expressing LSK cells in the spleens of tumor-bearing mice. (A) Endogenous GM-CSF expression in freshly isolated LSK cells and GMPs (FcγRhiCD34+ LK) (n = 6 per group). ***P < 0.001, by 2-way ANOVA followed by Dunnett’s test. (B) Immunoblot for NF-κB p65 and MAPK p38 activation in LSK cells isolated from BM and spleens of Hepa mice. p, phosphorylated; t, total. (C) Clonogenic ability of 500 BM or splenic LSK cells isolated from Hepa mice (n = 6 per group) in the methylcellulose-based assay. Anti–GM-CSF (αGM-CSF): 3 μg/ml. ***P < 0.001, by 2-way ANOVA followed by Dunnett’s test. (D) LSK cells were CFSE stained and cultured for 5 days in serum-free medium supplemented with SCF and the indicated concentration of anti–GM-CSF Abs in the cultures. The proliferation and differentiation HSPCs into myeloid cells are shown by CFSE dilution. (E) GM-CSF expression in LSK cells was examined after 24 hours of the cultures described in D. Numbers in the flow cytometric plots indicate the proportions of gated cells (A, D, and E). Results are shown as the mean ± SEM for mice in each group (A and C). Data are from 2 experiments (A), 3 experiments with cells pooled from 6 to 10 mice (C), or representative of at least 3 experiments (B, D, and E).
To confirm the function of endogenous GM-CSF, we found that neutralizing Abs against GM-CSF impaired CFU-GM activity (with stem cell factor [SCF], IL-3, IL-6, and erythropoietin in the methylcellulose-based culture) of splenic, but not BM, LSK cells (Figure 2C). In addition, neutralizing the endogenous GM-CSF in an essential HSPC culture condition (serum-free medium supplemented with SCF only) attenuated the proliferation of splenic LSK cells and their differentiation into myeloid cells in a dose-dependent manner (Figure 2D). These data suggested an important role of this endogenous signal in mediating LSK cell myelopoiesis in both simple and complex cytokine environments. Moreover, the presence of anti–GM-CSF Abs in the culture also attenuated the intracellular GM-CSF expression of these splenic LSK cells, suggesting a positive feedback of this endogenous GM-CSF signal (Figure 2E). Together, these findings indicate that a significant fraction of splenic LSK cells upregulates the GM-CSF expression, thereby potentiating their myeloid differentiation, which may represent a distinct stress-induced HSPC response in tumor-bearing mice.
Endogenous GM-CSF drives splenic HSPCs to generate myeloid suppressors. We next investigated whether and how this splenic HSPC response contributed to the immunosuppressive function of tumor-associated myeloid cells. To evaluate the overall effect of splenic EMH, we first performed splenectomy. We found that splenectomy did not change the frequencies or distribution of tumor myeloid cells (Supplemental Figure 3, A and B). However, it did abolish the suppressive activity of tumor CD11b+Ly6G+Ly6Clo polymorphonuclear-like (PMN-like) cells (or tumor PMN-MDSCs) (Figure 3, A and B), which was the major suppressive MDSC subset in Hepa tumors (Supplemental Figure 3, C and D). The abrogation of splenic EMH by splenectomy impaired the suppressive effect of tumor-infiltrating PMN-like cells on the proliferation and antigen-induced cytotoxic activity of T cells (Figure 3, A and B). We observed that splenectomy suppressed the expression of arginase 1 (Arg1) in tumor-infiltrating PMN-like cells (Supplemental Figure 3, E–G; see complete unedited blots in the supplemental material), which was associated with the inhibition of STAT3 activation (Supplemental Figure 3G). In addition, splenectomy increased the frequencies of tumor-infiltrating IFN-γ+CD3+CD8+ cytotoxic T lymphocytes (CTLs) by 2-fold (Figure 3C). These findings suggested an important role of splenic EMH in mediating the suppressive myeloid response.
Endogenous GM-CSF drives splenic HSPCs to produce myeloid suppressor cells. (A and B) Immunosuppressive effect of tumor-infiltrating CD11b+Ly6G+Ly6Clo PMN-like myeloid cells on anti-CD3– and anti-CD28–stimulated T cell proliferation (A) and on the cytotoxic activity of CD8+ T cells elicited by antigen-specific stimulation in vitro (B). Hepa mice were subjected to sham surgery or splenectomy (SPx). **P < 0.01 and ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (C) Frequencies of tumor-infiltrating IFN-γ+CD3+CD8+ CTLs in Hepa mice subjected to sham surgery or splenectomy. ***P < 0.001, by Student’s t test. (D and E) Splenic LSK cells were cultured in serum-free medium with SCF for 4 days. The Gr-1+ myeloid descendants were isolated and tested for their suppressive effect on anti-CD3– and anti-CD28–stimulated T cell proliferation at a ratio of 1:4 (D) and on the antigen-induced cytotoxic activity of CD8+ T cells (E). ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (F) Cartoon depicting the adoptive transfer of CD45.1- BM or splenic LSK cells into the spleens of CD45.1+ tumor-free recipient mice. (G–I) Representative phenotype (G) and suppressive activity (H and I) of donor-derived CD11b+Gr-1+ myeloid cells retrieved from the spleens of recipient mice (n = 3 per group) as described in F. **P < 0.01 and ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. Results are shown as the mean ± SEM (A–C, E, H, and I). CFSE+ splenocyte proliferation when cocultured with myeloid descendant cells at the indicated ratios (solid lines) or cultured alone (shaded) is shown (A, D, and H). Numbers in the flow cytometric plots indicate the proportions of gated cells (A, D, G, and H). Data are representative of 2 experiments (A–C, n = 3 per group in each experiment) or 3 experiments, with cells pooled from 6 to 10 mice (D–I).
GM-CSF is a critical regulator of MDSC generation and activation (8, 19). However, it has been established that de novo induction of a naive progenitor/precursor cell to generate fully functional MDSCs requires at least 2 signals (31, 32). GM-CSF alone failed to induce BM progenitors from Hepa mice to produce functional CD11b+Gr-1+ MDSCs in vitro (Supplemental Figure 3, H and I), consistent with a previous report (17). In contrast, GM-CSF effectively induced splenic LSK and GMP cells to generate myeloid cells with potent immunosuppressive activity in both serum-free and complete media (Supplemental Figure 3, H and I). These data suggested that the splenic HSPCs from Hepa mice, but not their BM phenotypic counterparts, included “primed” progenitors of myeloid suppressor cells. Given this finding, we tested whether the endogenous GM-CSF produced by LSK cells was sufficient for the generation of MDSCs. As expected, splenic LSK cells from Hepa mice generated highly suppressive myeloid descendants, without exogenous supplementation with GM-CSF, which could be completely prevented by the inclusion of anti–GM-CSF Abs in the culture (Figure 3, D and E). We consistently, obtained similar results using splenic LSK cells isolated from mice with DEN and CCl4-induced hepatoma and _Apc_Min/+ mice (Supplemental Figure 3, J and K).
Given the distinct capability of splenic HSPCs to produce and respond to GM-CSF, we speculated that these primed progenitors could readily generate myeloid suppressors in vivo, even independently of the presence of tumors. To test this hypothesis, we transferred CD45.1– splenic or BM LSK cells from Hepa mice into the spleens of naive CD45.1+ recipients. Five days after the adoptive transfer, we isolated donor cells and tested for their immunosuppressive activity (Figure 3F). Consistent with the in vitro findings discussed above, only the CD45.1–CD11b+Gr-1+ myeloid descendant cells of splenic LSK cells, but not those derived from BM, could effectively inhibit anti-CD3– and anti-CD28–stimulated proliferation and antigen-induced cytotoxic activity of T cells (Figure 3, G–I). These ex vivo data, together with the findings of endogenous GM-CSF expression and the effects of neutralizing Ab, indicate that the splenic HSPCs in Hepa mice include primed progenitors of potent myeloid suppressors that can readily respond to the endogenous GM-CSF signal and support the suppressive myeloid response.
Spleens of tumor-bearing mice recruit BM-derived circulating HSPCs. To investigate the underlying mechanism regulating the distinct properties of splenic LSK cells in Hepa mice, we first determined the cellular origin of these splenic HSPCs. The splenic HSPC population could be originated from the BM and/or the clonal expansion of local residents. To determine the original source(s) of these accumulated HSPCs, we generated tumor-bearing parabiotic pairs by joining WT B6 and EGFP+ mice (Supplemental Figure 4A). After 4 weeks of parabiosis, the tumor dramatically induced partner-derived (EGFP+) HSPC accumulation in the spleens of B6 mice (Supplemental Figure 4, B and C), suggesting circulating HSPCs compartment as an important source of splenic HSPCs. Importantly, removal of the spleen of the EGFP+ partner barely altered the number, composition, or CFU in culture (CFU-C) activity of the partner-derived splenic HSPCs, excluding the possibility that a recirculating splenic resident cell population was the major source. In addition, the spleens of tumor-bearing mice showed a 2-fold greater capacity for recruiting circulating BM-derived CFU-GM progenitors (Supplemental Figure 4, D and E). Thus, these results suggested that the majority of splenic HSPCs in Hepa mice were derived from the circulating cell population that originated from BM.
The splenic niche modulates LSK HSPC functional properties. Since the majority of splenic HSPCs are BM derived, we sought to investigate the mechanism(s) accounting for their distinct functions compared with those of their counterparts in BM. We reasoned that this difference could possibly result from the local “education” by the cancer-conditioned splenic niche and/or the selective recruitment of circulating HSPCs.
We first tested the “local education” hypothesis by coculturing naive BM LSK HSPCs with BM or splenic stromal cells isolated from control or Hepa mice (Figure 4A). The results showed that only the splenic stromal cells from Hepa mice could induce an increase in the CFU-GM activity (Figure 4B) and endogenous GM-CSF expression (Figure 4C) of BM-derived naive LSK cells. To determine whether splenic stromal cells could alter the capability of these naive early HSPCs in vivo, we adoptively transferred naive BM LSK cells into the spleens of control or Hepa mice (Figure 4D). We observed that splenic stroma of Hepa mice, but not that of control mice, induced these early progenitors to produce highly suppressive myeloid descendants in vivo (Figure 4E). These data indicate that the cancer-conditioned splenic niche is capable of educating naive LSK cells to generate myeloid immune suppressors.
Splenic stroma of tumor-bearing host supports LSK cells in the production of MDSCs. (A) Scheme of the coculture experiments. (B and C) CFU-C activity (B) and endogenous GM-CSF expression (C) of BM-derived naive LSK cells after coculture with CD45– stromal cells from the indicated tissues. ***P < 0.001, by 2-way ANOVA followed by Dunnett’s test. (D) Cartoon depicting the adoptive transfer of CD45.1– BM-derived naive LSK cells into the spleens of CD45.1+ tumor-free or tumor-bearing recipient mice. (E) Suppressive activity of donor-derived CD11b+Gr-1+ myeloid cells retrieved from the spleens of the recipient mice described in D. ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (F and G) CFU-C activity (F) and endogenous GM-CSF expression (G) of BM-derived naive LSK cells after coculture in Transwells with splenic stromal cells from Hepa mice with the indicated Abs (1 μg/ml) in the cultures. ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (H) After 4 days of coculture in Transwells as described in F and G, Lin– HSPCs were isolated and transferred into serum-free medium supplemented with SCF only. After another 3 days of culture, the Gr-1+ myeloid descendants were isolated and tested for their suppressive activity. CFSE+ splenocyte proliferation when cocultured with myeloid descendant cells at the indicated ratios (solid lines) or cultured alone (shaded) is shown (E and H). Numbers in the flow cytometric plots indicate the proportions of gated cells (C, E, G, and H). Data are shown as the mean ± SEM of 3 experiments, with cells pooled from 6 to 8 mice (B, E, and F) or are representative of 3 experiments, with cells pooled from 6 to 8 mice (C, G, and H).
This education effect was retained in the Transwell culture system (Figure 4, F–H), suggesting that this effect was mediated by soluble factor(s). We found that IL-6 was upregulated in the splenic stromal cells of Hepa mice (Supplemental Figure 4, F and G). The inclusion of anti–IL-6–neutralizing Abs in the Transwell assays attenuated the CFU-GM activity (Figure 4F) and GM-CSF expression (Figure 4G) of LSK cells cultured with Hepa splenic stromal cells and impaired the suppressive function of their myeloid descendants (Figure 4H). In support of this, recombinant mouse IL-6 induced GM-CSF expression in LSK cells (Supplemental Figure 4H). Together, these data demonstrate that splenic stromal cells of tumor-bearing mice can induce LSK cell functional alteration via soluble factors such as IL-6.
Upregulation of CCL2/CCR2 signaling in the spleen. Having revealed the role of the local education, we next investigated whether a selective recruitment mechanism might also contribute to this splenic HSPC response. Interestingly, we observed higher chimerisms in both splenic LSK and LK cells in Hepa tumor–bearing parabiotic pairs (Supplemental Figure 5A), suggesting a faster turnover of splenic HSPCs, which contradicted their accumulation. To further define the kinetics of HSPC turnover in the spleen, we surgically separated pairs of mice after 4 weeks of parabiosis. After separation, splenic HSPC chimerisms in tumor-free pairs were unchanged during the 96-hour observation, whereas the chimerisms in tumor-bearing mice significantly decreased (Supplemental Figure 5B). These results indicate a cancer-driven increase in splenic HSPC turnover, possibly due to their faster differentiation, since the apoptotic rates of splenic HSPCs were similar between the control and Hepa mice (Supplemental Figure 5, C–E). Thus, we reasoned that the accumulation of splenic HSPCs in Hepa mice might rely on the cancer-endowed stronger attraction for BM-derived circulating progenitor cells (Supplemental Figure 4E), thus outstripping the accelerated HSPC consumption.
To probe the mechanism involved in the recruitment of circulating HSPCs into the spleen, we examined the expression profiles of cytokines and chemokines (Supplemental Figure 6A). The CXCR4 ligand CXCL12 (also known as stromal cell–derived factor 1) is not only a critical mediator for HSPC recruitment and retention in BM, but also mediates splenic EMH induced by myeloablation, blood loss, or pregnancy (33). We were surprised to find that CXCL12 was downregulated in the spleens of tumor-bearing mice (Figure 5A and Supplemental Figure 6B). In contrast, the CCR2 ligand CCL2, along with the CXCR2 ligands CXCL2 and CXCL5, was among the most upregulated chemokines in the spleens of Hepa mice (Figure 5B and Supplemental Figure 6C). Since CXCR2 was not expressed on BM or splenic HSPCs (Supplemental Figure 6D), we focused on the CCL2/CCR2 axis in our study. To determine the potential source(s) of splenic CCL2, we analyzed its production in different splenic cell populations. Splenic stromal cells, especially VE-cadherin+ endothelial cells, markedly increased CCL2 production in Hepa mice (Figure 5C and Supplemental Figure 6E). We then analyzed the expression of the corresponding chemokine receptor CCR2 on HSPCs and found that a remarkably larger proportion of splenic LSK HSPCs from Hepa mice expressed CCR2 compared with those from BM or control spleen (Figure 5D).
Enhanced CCL2/CCR2 signaling in the spleens of Hepa mice. (A and B) Splenic Cxcl12 (A) and Ccl2 (B) mRNA expression levels. Values are relative to Actb mRNA expression (n = 6 per group). **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s test. (C) CCL2 release was measured by ELISA in the supernatants of 48-hour cultures of the indicated cell populations isolated from the spleens of control or Hepa mice (n = 3 per group, evaluated in triplicate). ***P < 0.001, by 2-way ANOVA followed by Bonferroni’s test. (D) Flow cytometric analysis of surface CCR2 expression on BM and splenic LSK and GMP cells from control (n = 7) and Hepa mice (n = 6). Numbers in the cytometric plots indicate the proportions of the gated cells. ***P < 0.001, by 2-way ANOVA followed by Tukey’s test. Data are representative of at least 2 experiments and presented as the mean ± SEM of the mice in each group (A–D).
Role of the CCL2/CCR2 axis in the splenic recruitment of myeloid HSPCs. To investigate the role of the CCL2/CCR2 axis in mediating splenic recruitment of circulating myeloid progenitors, we measured CFU-GM homing. RS 504393, a highly selective CCR2 antagonist, effectively reduced the splenic recruitment of circulating CFU-GM progenitors in tumor-bearing mice to basal levels (Figure 6A). To corroborate the direct action of CCL2 on HSPCs, we transferred CCR2-deficient (EGFP–) and CCR2-competent (EGFP+) BM cells simultaneously into control (groups 1 and 3) or tumor-bearing (groups 2 and 4) recipients (Figure 6B). Irrespective of the CCR2 expression status in recipient mice, the ratios of CCR2–/–EGFP– to CCR2+/+EGFP+ CFU-GM progenitors homing to the spleens of tumor-bearing hosts were at least 3-fold lower compared with the ratios of input and those in control spleens (Figure 6C).
The CCL2/CCR2 axis mediates the enhanced splenic recruitment of circulating HSPCs in Hepa mice. (A) Percentages of transferred BM-derived CFU-GM progenitors homing to the spleens of control or Hepa mice. **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Tukey’s test. (B) Scheme of competitive homing assays. (C) Ratios of EGFP–CCR2–/– versus EGFP+CCR2+/+ BM-derived CFU-GM progenitors homing to the spleens of control or Hepa mice (n = 8 for CCR2+/+ recipient groups; n = 4 for CCR2–/– recipient groups), as described in B. ***P < 0.001, by 1-way ANOVA followed by Tukey’s test. ctrl, control. (D) Cartoon depicting the generation of control and Hepa-bearing parabiotic pairs. (E) Flow cytometric quantification of CD45.2+ partner-derived splenic LSK and GMP cells from the parabiotic mouse pairs described in D. ***P < 0.001, by 2-way ANOVA followed by Tukey’s test. (F) Quantification of BM and splenic LSK and GMP cells in CCR2+/+ or CCR2–/– Hepa mice. ***P < 0.001, by Student’s t test. (G) GM-CSF expression in splenic LSK cells from CCR2+/+ or CCR2–/– Hepa mice was measured by flow cytometry. *P < 0.05, by Student’s t test. (H) Immunosuppressive activity of tumor-infiltrating CD11b+Ly6G+Ly6Clo PMN-like cells. **P < 0.01 and ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (I) Frequencies of tumor-infiltrating IFN-γ+CD3+CD8+ CTLs in CCR2+/+ and CCR2–/– Hepa mice. **P < 0.01, by Student’s t test. Data are representative of 2 experiments and presented as the mean ± SEM. n = 3 per group in each experiment (A–E); n = 4 for the CCR2+/+ group, n = 5 for the CCR2–/– group in each experiment (F); n = 3 per group in each experiment (G–I).
Next, we tracked CCR2-deficient and CCR2-competent HSPCs in parabiotic pairs with or without Hepa tumors (Figure 6D). The proportion of CCR2-competent, partner-derived (CD45.2+) splenic HSPCs was significantly increased in the tumor-bearing pairs. As expected, this substantial elevation in the partner-derived HSPC proportion was abrogated if partner-derived circulating progenitor cells lacked CCR2 expression (Figure 6E). Likewise, the tumor-elicited splenic accumulation of LSK and GMP cells was attenuated in CCR2–/– Hepa mice, while we observed little change in the BM (Figure 6F). These results indicate that CCL2-CCR2 interaction directly mediates the splenic recruitment of circulating HSPCs with myeloid potential in tumor-bearing hosts. More important, the remaining splenic LSK cells in CCR2-deficient Hepa mice had markedly decreased levels of endogenous GM-CSF expression (Figure 6G), suggesting that the education by the spleen required the peripheral CCR2+ LSK subset (34).
Accordingly, lack of CCR2 expression on HSPCs recapitulated the effect of splenectomy on the tumor-promoting myeloid response. The absence of the endogenous GM-CSF–driven splenic EMH impaired the suppressive activity of tumor PMN-like cells in CCR2-deficient Hepa mice (Figure 6H and Supplemental Figure 6F), which allowed an increase in the number of tumor-infiltrating IFN-γ+CD3+CD8+ CTLs (Figure 6I). Although knockout of CCR2 also reduced the tumor-infiltrating CD11b+Ly6G–Ly6Chi monocytic cells (Supplemental Figure 6G), this was supposed to be a minor effect compared with the functional alteration of PMN-like cells, given the relatively smaller number and less suppressive ability of these cells in the Hepa model (Supplemental Figure 3, A–D). Thus, these findings indicate that the recruitment of HSPCs via the CCL2/CCR2 axis, followed by the splenic education, is an integral and essential process for inducing the distinct splenic EMH that promotes the systemic myeloid response.
Abrogation of splenic EMH synergistically enhances anti–PD-L1 therapy. Given the above findings that the abrogation of splenic EMH enhanced antitumor immunity (Figure 3), we expected that it might potentially promote immunotherapy. Hepa mice were treated with splenectomy or sham surgery and sacrificed when the tumor reached a diameter of approximately 1.5 cm. In Hepa mice inoculated with 7.5 × 105 tumor cells, the impact of splenectomy on mouse survival was mild but statistically significant (Figure 7A). The effect of splenectomy was more robust when the initiating tumor cells were reduced (Supplemental Figure 7A). This agrees with previous reports suggesting that the impact of splenectomy on cancer progression is dependent on the tumor settings in animal models and is conditional in patients (21, 35–37). We noticed that splenectomy did not decrease the expression of PD-L1 on tumor PMN-like cells (Supplemental Figure 3F), which might indicate an opportunity for combined therapy with anti–PD-L1 (38, 39). Monotherapy of anti–PD-L1 treatment did not affect the number or phenotype of splenic HSPCs (Supplemental Figure 7, B-D) and only elicited marginal therapeutic effects in Hepa mice (Figure 7A) and in another hepatoma mouse model based on a BALB/c background (Supplemental Figure 7, E and F). However, splenectomy synergistically enhanced the therapeutic efficacy of anti–PD-L1 treatment (Figure 7A and Supplemental Figure 7F). Complete regression of established cancer was achieved in over 50% of Hepa mice. These results suggest a potential role for targeting splenic EMH to enhance immunotherapy.
Abrogation of splenic EMH synergistically enhances anti–PD-L1 efficacy. (A) Survival of Hepa mice subjected to sham surgery or splenectomy with or without anti–PD-L1 treatment. *P < 0.001 and ***P < 0.001, by log-rank test compared with the sham group; ###P < 0.001, by log-rank test compared with the splenectomy group; †††P < 0.001, by log-rank test compared with the sham plus anti–PD-L1 group. (B) Fold changes in the numbers of splenic LSK and GMP cells in Hepa mice. ***P < 0.001, by 2-way ANOVA followed by Dunnett’s test. (C) Proportions of GM-CSF+ splenic LSK cells in Hepa mice. ***P < 0.001, by 2-way ANOVA followed by Dunnett’s test. (D–F) Immunosuppressive activity of tumor-infiltrating CD11b+Ly6G+Ly6Clo PMN-like cells (D and E) and the frequencies of tumor-infiltrating IFN-γ+CD3+CD8+ CTLs (F). Hepa mice were treated with low-dose sorafenib (Sora) 7 days prior to examination or were left untreated as controls. ***P < 0.001, by 2-way ANOVA with Bonferroni’s correction. (G) Survival of Hepa mice that received no treatment or treatment with low-dose sorafenib or anti–PD-L1 or both. For some groups, mice received adoptive transfers of 1 × 106 splenic HSPCs or 5 × 106 MDSCs 3 and 10 days after the latter dose of sorafenib. ***P < 0.001, by log-rank test compared with the isotype plus sorafenib group; ###P < 0.001, by log-rank test compared with the anti–PD-L1 group; †††P < 0.001, by log-rank test compared with the anti–PD-L1 plus sorafenib group. (H) Survival of CCR2+/+ or CCR2–/– Hepa mice treated with anti–PD-L1 or isotype Abs. ***P < 0.001, by log-rank test compared with the CCR2–/– plus isotype group; #P < 0.05, by log-rank test compared with the CCR2+/+ plus anti–PD-L1 group. Data are representative of 2 experiments (B–F; n = 3 per group in each experiment) and indicate the mean ± SEM, or were pooled from 2 experiments (n = 7–11 per group as indicated in A, G, and H).
In order to preserve the normal physiological functions of the spleen while inhibiting splenic EMH, we tested whether selectively targeting splenic HSPCs could also synergize with immunotherapies. Low-dose sorafenib, a c-Kit inhibitor (40), induced the apoptosis of splenic HSPCs (Supplemental Figure 7G), inhibited their proliferation (Supplemental Figure 7H), and reduced the numbers of HSPCs in spleen but had much less effect on those in the BM (Figure 7B and Supplemental Figure 7I). Moreover, low-dose sorafenib treatment attenuated endogenous GM-CSF expression in splenic LSK cells (Figure 7C). Consistent with splenectomy, low-dose sorafenib inhibited the suppressive effects of tumor PMN-MDSCs on T cell proliferation and cytotoxic activity (Figure 7, D and E), increased the frequencies of infiltrating IFN-γ+CD3+CD8+ CTLs (Figure 7F), and recapitulated the synergistic effect when combined with PD-L1 blockade (Figure 7G). Importantly, as shown in Figure 7G, this synergistic effect was abrogated by restoring the splenic Linlo/–c-Kithi population including LSK and downstream myeloid progenitor cells, which are capable of generating myeloid suppressor cells (Supplemental Figure 3, H and I), but not by transferring splenic MDSCs, possibly because of their short lifespan (11). These data demonstrate the important role of splenic EMH in impeding antitumor immunity and immunotherapy. Likewise, anti–PD-L1 treatment elicited profound therapeutic responses in CCR2-deficient Hepa mice (Figure 7H). Together, these findings demonstrate that targeted therapies against splenic EMH can effectively synergize with immune checkpoint blockade and thus may represent a novel strategy to enhance currently available immunotherapy.
Splenic EMH in patients with different types of solid tumors. Having established the function, mechanism, and significance of tumor-induced splenic EMH in mouse models, we sought to assess the clinical relevance of splenic EMH. CD133 is a marker for human HSPCs (41). We confirmed that splenic CD133+ cells had a Lin–CD34hi phenotype (Supplemental Figure 8A) and were highly enriched for CFU-GM activity (Supplemental Figure 8B). We examined the frequencies of splenic HSPCs in situ by staining for CD133 in formalin-fixed, paraffin-embedded splenic tissues from 135 patients with different types of solid tumors and 34 noncancer patients (Supplemental Table 1). The frequencies of splenic CD133+ cells in the patients with cancer were significantly higher than those in patients without cancer (Figure 8, A and B, and Supplemental Figure 8C). The densities of CD11b+ myeloid cells were also markedly increased in the spleens of cancer patients (Figure 8, A and C), which significantly correlated with the splenic CD133+ HSPC levels (Figure 8D).
Splenic EMH in patients with different types of solid tumors. (A) Representative immunohistochemical staining of CD133+ and CD11b+ cells in splenic specimens from a total of 135 patients with hepatocellular carcinoma (HCC; n = 22), gastric cancer (GC; n = 86), kidney cancer (KC; n = 16), or pancreatic cancer (PC; n = 11) and from 34 noncancer patients with splenic hemangioma (n = 6) or cirrhosis (n = 28). Red arrows point to the CD133+ cells in the fields. Scale bars: 50 μm. (B and C) Quantification of CD133+ HSPCs (B) and CD11b+ myeloid cells (C) in the splenic specimens described in A. ***P < 0.001, by Kruskal-Wallis test. (D) Correlation between splenic CD133+ HSPC and splenic CD11b+ myeloid cell densities. The results of a Spearman’s rank correlation analysis are indicated. (E) Patients with gastric cancer were divided into 2 groups according to the median value of the splenic CD133+ cell frequencies. The duration of overall survival was estimated using the Kaplan-Meier method and compared by log-rank test (P = 0.0145).
Finally, we evaluated the clinical significance of cancer-induced splenic accumulation of HSPCs. Kaplan-Meier analyses revealed that the high frequency of CD133+ progenitors in the spleen negatively impacted the overall survival of patients with gastric cancer (P = 0.0145, hazard ratio [HR] = 2.096, 95% CI: 1.158–3.794) (Figure 8E). Patients with lower splenic CD133+ cell frequencies survived significantly longer (median, 29 months) than did patients with higher splenic CD133+ cell frequencies (median, 18 months). This analysis of patients with other types of tumors was not applicable yet, either because of an insufficient sample size or because the majority of the patients were recently diagnosed at the early stage (Supplemental Table 1). Overall, in line with our findings in mouse models, the clinical data presented so far suggest the functional and clinical importance of splenic EMH in patients with cancer.