Tumor cells disseminate early, but immunosurveillance limits metastatic outgrowth, in a mouse model of melanoma

Abstract

Although metastasis is the leading cause of cancer-related death, it is not clear why some patients with localized cancer develop metastatic disease after complete resection of their primary tumor. Such relapses have been attributed to tumor cells that disseminate early and remain dormant for prolonged periods of time; however, little is known about the control of these disseminated tumor cells. Here, we have used a spontaneous mouse model of melanoma to investigate tumor cell dissemination and immune control of metastatic outgrowth. Tumor cells were found to disseminate throughout the body early in development of the primary tumor, even before it became clinically detectable. The disseminated tumor cells remained dormant for varying periods of time depending on the tissue, resulting in staggered metastatic outgrowth. Dormancy in the lung was associated with reduced proliferation of the disseminated tumor cells relative to the primary tumor. This was mediated, at least in part, by cytostatic CD8+ T cells, since depletion of these cells resulted in faster outgrowth of visceral metastases. Our findings predict that immune responses favoring dormancy of disseminated tumor cells, which we propose to be the seed of subsequent macroscopic metastases, are essential for prolonging the survival of early stage cancer patients and suggest that therapeutic strategies designed to reinforce such immune responses may produce marked benefits in these patients.

Figure 1. Unique kinetics of tumor onset in various tissues and organs.

The onset of superficial tumors was determined by biweekly clinical examination. Superficial tumors rarely affected mouse viability; therefore, mice bearing such tumors could be analyzed by necropsy at early and late time points for the presence of internal tumors. Kaplan-Meier curves were generated from the analysis of 180 untreated RET.AAD mice.

Figure 2. Pattern of genetic alterations detected by GW-SNP profiling.

(A) Eye tumors (E) and non-eye tumors (NE) displayed similar numbers of total mutations. (B) Eye tumors and non-eye tumors displayed a similar percentage of unique mutations. (C) Percentage of eye mutations that were conserved in the other tumors. The reference tumor is either the left eye (LE) or the right eye (RE) tumor. (A–C) Horizontal bars denote medians. (D) Overall percentage of mutations in the reference tumor that were conserved in the other tumors from the same mouse (n = 4). Reference tumors were either eye or non-eye. Data represent mean ± SEM.

Figure 3. Tumors cluster according to mice.

Tumors (n = 26) taken from 5 mice were clustered according to their pattern of mutations detected by GW-SNP profiling (see Supplemental Figure 4). Distance matrix (A) or PCA (B) showed that tumors from the same mouse tended to cluster together. Colored symbols in B correspond with mice color-coded as in A. (C) Phylogram of the 9 tumors from mouse 3. The number of mutations conserved along each horizontal branch is indicated in red. The number of mutations unique to each tumor is shown in blue. The tissue origin of other tumors is given in Supplemental Table 2.

Figure 4. Dct expression in lungs correlates with the presence of S100B⁺ cells.

Lungs of RET.AAD mice (n = 34) or ret^–/–AAD littermates (n = 32) were analyzed for the presence of S100B⁺ cells by IHC. Dct expression was measured in the contralateral lung by qRT-PCR. (A–C) Representative examples of S100B staining of lungs from ret^–/–AAD mice (A) or from RET.AAD mice, either with individual DTCs or micrometastases only (B) or with macroscopic nodules (C). Arrows indicate DTCs or nodules. Scale bars: 100 μm. (D) Dct expression was measured by qRT-PCR in lungs of RET.AAD mice either with individual DTCs and/or micrometastases (DTC/Micro) or with macrometastases (Macro). Nontransgenic littermates (ret^–/–) were used as controls. Dct expression is shown as the log₂ ratio of Dct over GAPDH. Dotted line represents mean expression in ret^–/– lungs + 3 SD. Expression in excised macroscopic nodule (Tumor) is also shown for comparison.

Figure 5. Tumor cell dissemination to internal organs starts at 3 weeks of age.

Dct expression was measured by qRT-PCR in the lymph nodes, lungs, heart, kidney, liver, stomach, colon, bone marrow, thymus, bladder, and small intestine (n = 11 organs per mouse) collected from RET.AAD (RET^+/–) mice aged 1 week (n = 3), 3 weeks (n = 3), and 5 weeks (n = 4). As controls, 11 age-matched nontransgenic littermates (ret^–/–) were used.

Figure 6. Morphology of eye tumors at various ages.

Eye sections from 2- (A), 4- (B and C), 5- (D), 6- (E), and 50-week-old (F) mice. (A–C) Black arrows indicate hyperplastic lesions within the choroid (Ch). The white arrow marks a round metastatic nodule attached to the sclera (Sc). Re, retina. (D–F) Note the multinodular morphology of the tumors. Images are representative of more than 20 analyzed eye tumors. Scale bars: 50 μm (A and B); 300 μm (C–F).

Figure 7. Eye metastases have a higher mitotic index than do lung tumors of similar size.

Eye (A) and lung (B) sections were stained for tumor cells (S100B; red) and the proliferation marker Ki-67 (blue). Scale bars: 100 μm. (C) Number of proliferating tumor cells (S100B⁺Ki-67⁺) as a function of tumor size in eye (circles) and lung (squares) tumors. Data are from 12 eyes and 17 lungs from 22 mice, representing a total of 103 tumors.

Figure 8. CD8 depletion accelerates the development of visceral tumors, but has no effect on cutaneous tumors.

(A) Development of visceral metastases was followed by PET scan 1 month (M1) and 2 months (M2) after initiation of the depletion of CD8⁺ T cells (n = 10). Control mice (n = 9) were injected with rat IgG control. Data represent mean ± SEM. (B–D) Typical images of genital (B) or lung tumors (C and D). Scale bars: ~1 cm. (E) Skin tumors were detected at necropsy. Results are pooled from 2 independent experiments.

Figure 9. CD8⁺ T cells control tumor cell proliferation.

Lungs of CD8-depleted (Treated) or control mice were analyzed by IHC for the presence of individual (A) or clustered (B) S100B⁺ cells. (C) The number of proliferating cells in each tumor was measured by 2-color IHC using Ki-67– and S100B-specific antibodies. (D) The area of lung tumors was measured using Image-Pro. Results are from 9 control and 11 treated mice in 2 independent experiments, representing a total of 81 and 64 tumors, respectively. Data represent mean ± SEM.