Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery

Abstract

Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b(+)F4/80(hi)CD206(hi) and CD11b(+)F4/80(hi)CD124(hi)) macrophages and CD4(+)Foxp3(+) regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.

Fig. 1.

Vaccination of primary tumors and adjuvant vaccination of recurrent tumors yields differential efficacy. (A) Ad.E7 vaccination of mice bearing primary TC1 tumors (at day 7 and 14) resulted in decreased tumor growth and increased overall survival. (B) Vaccination of mice bearing recurrent TC1 tumors (at postoperative day 4 and 11) did not correlate with decreased growth of recurrent tumors or survival benefits. (C) Flow cytometry of representative spleen and tumors of mice undergoing Ad.E7 vaccination demonstrates that epitope-specific T cells are generated equally in primary and recurrent disease scenarios. (D) Bar graph representation of the number of E7-specific CD8 T cells in spleens (Left) and tumors (Right) of mice bearing primary and recurrent tumors. (E) Immunohistochemistry of representative tumors reveals that recurrent tumors have less CD8 T-cell trafficking than primary tumors. (F) Tumor neutralization assay demonstrating that splenic CD8 T cells harvested from vaccinated mice with primary or recurrent tumors are equally capable of neutralizing in vivo TC1 tumor cells. As a control, TC1 tumor cells were injected alone or with CD8 T cells harvested from spleens of tumor-naive mice. *P < 0.05; **P < 0.01.

Fig. 2.

Splenic immunocytes are similar in primary and recurrent tumor states. (A) By flow cytometry, the number of MDSCs (CD11b⁺Gr⁺) was comparable in mice bearing primary and recurrent tumors. Furthermore, the MDSC percentage of total splenic leukocytes (CD45⁺) was similar in primary and recurrent tumors. The ratio of splenic MDSCs to CD8⁺ T cells was not statistically different in primary and recurrent tumors. (B) A similar number of Tregs (CD4⁺Foxp3⁺) was found in spleens of mice bearing primary and recurrent TC1 tumors. Foxp3⁺ Tregs accounted for a similar proportion of total splenic leukocytes and total CD4 T cells. (C) The absolute number of splenic CD8 T cells and the percentage of CD8 T cells of splenic leukocytes were not statistically different in mice bearing primary and recurrent tumors. (D) In vivo tumor neutralization assay demonstrating that splenic CD8 T cells obtained from mice bearing primary and recurrent tumors are equally capable of inhibiting the growth of TC1 tumors in naive mice. As a control, TC1 tumor cells were injected alone or with CD8 T cells harvested from the spleens of tumor-naive mice. **P < 0.01.

Fig. 3.

Immunosuppressive TAMs infiltrate recurrent tumors. (A) Flow cytometry shows that similar absolute numbers of leukocytes (CD45⁺) infiltrate primary and recurrent tumors; the majority are macrophages (CD11b⁺Ly6G-F4/80+). (B) Macrophages express higher levels of CD206 in recurrent tumors than in primary tumors. (C) Macrophages also express higher levels CD124 in recurrent tumors than in primary tumors. (D) Selective depletion of macrophages using liposomal encapsulated clodronate decreases growth in recurrent tumors but has no significant effect on primary tumors. Arrows indicate time of clodronate administration. **P < 0.01. (E) Flow cytometry confirmed depletion of F4/80 macrophages.

Fig. 4.

Tregs infiltrate recurrent tumors and are increasingly prevalent in lymph nodes draining recurrent tumor. (A) Tregs (CD4⁺Foxp3⁺) accounted for a larger percentage of total tumor cells in recurrent tumor states. However, the percentage of Tregs of intratumoral CD4⁺ T cells remained constant in mice bearing primary and recurrent tumors. (B) Tregs accounted for a larger percentage of total leukocytes in lymph nodes draining recurrent tumors than in lymph nodes draining primary tumors. Furthermore, the ratio of Tregs to total CD4⁺ T cells was markedly increased in the lymph nodes draining recurrent tumors. (C) Selective depletion of CD4 T cells using anti-CD4 antibodies did not affect the growth of primary tumors but significantly inhibited the growth of recurrent tumors. Arrows indicate time of administration of anti-CD4 antibody. **P < 0.01. (D) Using immunohistochemistry, we confirmed that there were fewer CD8 T cells trafficking into recurrent tumors than into primary tumors.

Fig. 5.

Recurrent tumors have a pronounced change in the intratumoral cytokine milieu. (A) TC1 flank tumors were harvested at 0, 2, 6, 24, and 48 h, and lysates were analyzed by Luminex and ELISA. (B) SM16 and rofecoxib were administered in the chow for 5 d perioperatively (area highlighted in red) to mice bearing flank TC1 tumors. Flow cytometry of (C) FoxP3 and (D) CD206 and CD124 was done on harvested flank tumors after inhibition of TGF-β or COX2.

Fig. 6.

Elimination of Tregs and macrophages restores adjuvant vaccine efficacy. (A) The growth of recurrent TC1 tumors was decreased significantly in mice that were randomized to Ad.E7 vaccination with CD4 depletion (Combo) as compared with mice receiving either Ad.E7 or CD4-depleting antibodies alone. (B) By flow cytometry, the combination of Ad.E7 vaccination with CD4 depletion generated an increase of CD8 T cells specific to the E7 antigen in the spleen and tumor. (C) Combining vaccination with CD4 T cell depletion increased the percentage of CD8 T cells secreting IFN-γ after 6 h of in vitro stimulation with PMA/ionomycin (Bottom). (D) Immunohistochemistry of representative recurrent tumors show that Ad.E7 combined with CD4-depleting antibodies also increased the number of intratumoral CD8 T cells in recurrent lesions. *P < 0.05; **P < 0.01. (E) Mice bearing recurrent TC1 tumors that were randomized to Ad.E7 vaccination with macrophage depletion (liposomal clodronate) had significantly decreased recurrent tumor growth compared with mice receiving monotherapy.

Fig. P1.

(A) Vaccination of primary tumors and adjuvant vaccination of recurrent tumors yields differential efficacy. Ad.E7 vaccination of mice bearing primary TC1 tumors (on days 7 and 14) resulted in decreased growth and increased overall survival. Vaccination of mice bearing recurrent TC1 tumors (on postoperative days 4 and 11) did not correlate with decreased recurrence growth or survival benefits. (B) Immunosuppressive tumor-associated macrophages and Tregs dominate recurrent tumors. Macrophages in recurrent tumor microenvironments had increased CD206. Mice bearing recurrent TC1 tumors receiving Ad.E7 vaccination with CD4-depletion had significantly decreased recurrent tumor growth compared with those receiving Ad.E7 or CD4-depleting antibodies alone. *P < 0.05; **P < 0.01.