Research ArticleCANCER IMMUNOLOGY

The immune system profoundly restricts intratumor genetic heterogeneity

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Science Immunology  23 Nov 2018:
Vol. 3, Issue 29, eaat1435
DOI: 10.1126/sciimmunol.aat1435
  • Fig. 1 Lymphoma B cells developing in Eμ-myc transgenic mice display extensive intratumor genetic diversity.

    (A) Experimental setup. Lymphoma cells were isolated from the lymphoid organs of Eμ-myc transgenic mice. Metaphases were prepared, and 20 to 50 cells from each individual tumor were subjected to SKY. (B and C) Genetic heterogeneity in different cells isolated from the same tumor. (B) Representative images of various chromosomes obtained from four individual cells from one tumor. (C) Tables recapitulating the karyotypes of 40 cells analyzed from each individual tumor. Each table corresponds to the tumor cells isolated from one mouse. Red represents gain of chromosomes, black represents loss of chromosomes, and gray shows translocations. Data are representative of a total of five tumors isolated from male or female tumor-bearing Eμ-myc mice.

  • Fig. 2 A multicolor barcoding method to study intratumor clonal diversity.

    (A) Experimental setup used to produce color-barcoded tumors. Tumor cells isolated from a lymphoma-bearing Eμ-myc mouse were retrovirally transduced with a mixture of CFP- and GFP-encoding viruses or a mixture of GFP- and YFP-encoding viruses. CFP+, GFP+, YFP+, CFP+GFP+, and GFP+YFP+ cells were sorted, and a mixture containing an equal proportion of each cell population was prepared. Tg, transgenic; FITC, fluorescein isothiocyanate. (B) Tracking intratumor heterogeneity using a multicolor labeling strategy. Multicolor-barcoded cells were injected in individual mice. At various time points, the tumor composition was analyzed by flow cytometry and/or intravital imaging of the bone marrow.

  • Fig. 3 Anatomical and microanatomical clonal heterogeneity in B cell lymphoma development.

    (A) Color-barcoded lymphoma B cells isolated from a male Eμ-myc transgenic mouse were injected intravenously into male recipients. Tumor cellular composition was analyzed 3 weeks later in the bone marrow (BM), spleen, lymph nodes (LN), and nonlymphoid sites of tumor growth. Pie charts show the distribution of individual colored tumors in the indicated organs for three representative animals. (B) The graph displays the evenness index measured in different organs for 11 individual mice. ***P < 0.001 (one-way ANOVA with Bonferroni’s corrections). n.s., not significant. (C) Spatially organized architecture of clonal growth in the bone marrow. Color-barcoded tumor cells were injected intravenously, and recipients were subjected to intravital imaging of the bone marrow at the indicated time points. Two examples for each time points are shown and are representative of multiple regions of the bone marrow imaged in two independent experiments. Scale bar, 50 μm.

  • Fig. 4 CD8+ T cells can infiltrate the bone marrow to form contacts with tumor cells.

    (A to C) Color-barcoded lymphoma B cells isolated from a male Eμ-myc transgenic mouse were injected intravenously into female recipients. On day 10, activated CD8+ T cells bearing the anti–H-Y MataHari TCR were adoptively transferred. Intravital imaging of the bone marrow was performed 4 hours later. (A) Representative image showing CD8+ T cell infiltration of colored tumor patches. Areas in the white square were enlarged to illustrate T cell–tumor cell interactions. Note that T cell infiltration in the different tumor patches showed a degree of heterogeneity. (B) CD8+ T cells are preferentially confined within tumor patches. Tracks of CD8+ T cells are shown in magenta, and tumor areas are delimited in white dashed lines. (C) Representative time-lapse images showing T cell–tumor cell encounters and subsequent establishment of stable cellular interactions. (D to F) Lymphoma B cells isolated from a male Eμ-myc transgenic mouse were transduced with a FRET-based reporter for caspase 3 activity and were injected intravenously into female recipients. On day 10, activated CD8+ T cells bearing the anti–H-Y MataHari TCR (SNARF labeled) and similarly activated CD8+ T cells from GFP-expressing OT-I TCR transgenic mice were adoptively transferred. Intravital imaging of the bone marrow was performed 4 hours later. (D) Left: Representative image showing MataHari T cells (magenta), OT-I T cells (green), live tumor cells (white), and apoptotic tumor cells (blue). Right: Tracks of individual MataHari and OT-I T cells are shown, illustrating a higher confinement for MataHari T cells. Scale bar, 50 μm. (E) Graph depicts velocities of individual OT-I and MataHari T cells (n = 352 and n = 295, respectively). Data are from two representative movies. ***P < 0.001 (unpaired t test). (F) Examples of tumor cells undergoing apoptosis after interactions with MataHari T cells.

  • Fig. 5 The immune system strongly restricts tumor clonal heterogeneity.

    (A to D) Color-barcoded lymphoma B cells isolated from a male Eμ-myc transgenic mouse were injected intravenously into male or female recipients. (A) Tumor growth was monitored by measuring the percentage of tumor cells among total B cells in the blood at various time points. At least six mice were analyzed in each group. **P < 0.01 (unpaired t test). (B) Female recipients display a highly reduced intratumoral clonal heterogeneity compared with their male counterparts. Representative flow cytometry plots showing the tumor cell composition retrieved from the bone marrow of a male or a female recipient at 3 weeks. (C) Pie charts representing the tumor composition in the bone marrow of three representative male and female recipients. (D) Graph shows the evenness index calculated from tumor cells retrieved at 3 weeks from the bone marrow of male (n = 17) or female (n = 22) recipients. Data are pooled from three independent experiments. ***P < 0.001 (unpaired t test). (E and F) Progressive decrease in tumor heterogeneity in female recipients. Longitudinal analysis of tumor composition analyzed in the blood of a representative male or female recipient. (F) Kinetic analysis of the evenness index for color-barcoded tumor injected in male, female, or female Rag2−/− (Rag2 KO) mice and recovered in the blood. At least six mice were analyzed for each group. ***P < 0.001 and ****P < 0.0001 (one-way ANOVA with Bonferroni’s corrections).

  • Fig. 6 Mechanisms of immune-mediated restriction of intratumor heterogeneity.

    (A to C) Color-barcoded lymphoma B cells isolated from a male Eμ-myc transgenic mouse (Fig. 1C, lymphoma #1) were injected intravenously into male or female recipients. The injected cell population and the tumor cells recovered from the bone marrow of male or female recipients were subjected to SKY or flow cytometry. (A) The immune response restricts the genetic diversity of tumor cells developing in female recipients. Tables recapitulating the karyotypes of 31 cells analyzed from the tumors of two male and two female recipients. Red represents gain of chromosomes, black represents loss of chromosomes, and gray shows translocations. (B) Fluorescence intensities were analyzed by flow cytometry. Representative histograms from one male and one female show the fluorescence intensities of the GFP+ population (top) and YFP+ population (bottom). (C) Graph shows normalized median fluorescence (FITC channel for all the populations except for the CFP+ population for which AmCyan fluorescence is considered). The relative fluorescence is set to 1 for male recipients. Data are pooled from three independent experiments. *P < 0.05; **P < 0.01 (unpaired t test). A minimum of 18 mice were analyzed for each group. (D) Tumor cells expressing a fluorescent protein are eliminated in female but not in male mice. A mixture of fluorescent and unlabeled tumor cells was injected in male, female, or MataHari TCR transgenic Rag2−/− mice. After 3 weeks, tumors were isolated from the bone marrow, and the percentage of fluorescent tumor cells among tumor cells was analyzed by flow cytometry. Error bars represent SEM. Data are pooled from two independent experiments with a total of at least seven mice per group. *P < 0.05 (one-way ANOVA with Bonferroni’s corrections). (E) CD8+ T cells retrieved from the bone marrow of tumor-bearing recipients were restimulated for 4 hours with a GFP peptide (10 μM) in the presence of GolgiPlug and assayed for intracellular IFN-γ expression by flow cytometry. *P < 0.05 (one-way ANOVA).

  • Fig. 7 The immune system strongly restricts tumor genetic heterogeneity.

    Lymphoma B cells isolated from a male Eμ-myc transgenic mouse were injected intravenously into male or female recipients. Tumor cells recovered from the bone marrow or nonlymphoid sites of male or female recipients were subjected to whole-exome sequencing. (A and B) Higher intratumor genetic heterogeneity in tumors developing in the bone marrow as compared with nonlymphoid site in the same male animal. (A) The VAF for each SNV is shown with the corresponding chromosome. SNVs with VAFs <20% are highlighted in red. (B) Graph shows the density distribution of VAFs. (C to E) Higher intratumor genetic heterogeneity in tumors developing in male compared with female recipients. (C) Whole-exome sequencing was performed on eight tumor samples (recovered from the bone marrow of four male and four female recipients). For two representative tumors of each group, the VAF for each SNV is shown with the corresponding chromosome. SNVs with VAFs <20% are highlighted in red. (D) Graphs show the density distribution of VAFs. (E) After whole-exome sequencing, the MATH score was used to evaluate intratumor heterogeneity in tumors recovered from male (n = 4) and female (n = 4) recipients. *P < 0.05 (unpaired t test)

  • Fig. 8 Immune checkpoint inhibition reduces intratumor heterogeneity.

    (A to D) Lymphoma B cells isolated from a male Eμ-myc transgenic mouse were injected intravenously into male recipients. Recipients were injected every 3 days with the indicated Abs. (A) Pie charts representing the tumor composition in the bone marrow of three representative mice from each group at 3 weeks. (B) Graph shows the evenness index calculated from tumor cells retrieved at 3 weeks from the bone marrow of individual mice. Each dot represents one animal. Data are compiled from two independent experiments with at least 13 mice per group. *P < 0.05 (unpaired t test). (C) Combined anti–PD-1 and anti–CTLA-4 treatment reduces tumor diversity. Graph shows the evenness index calculated from bone marrow tumor cells for the indicated group. Each dot represents one animal. **P < 0.01 (unpaired t test). (D) Graph shows normalized median fluorescence (FITC channel for all the populations except for the CFP+ population for which AmCyan fluorescence is considered). The relative fluorescence is set to 1 for isotype-treated recipients. *P < 0.05 (unpaired t test). (E and F) v-abl–transformed pro-B cells from female mice were injected intravenously into female recipients. Recipients were injected every 3 days with the indicated Abs. (E) Graph shows percentage of CD8+ T cells present in the bone marrow at 2 weeks after tumor injection. Each dot represents one animal. *P < 0.05 (unpaired t test). (F) Graph shows the evenness index calculated for tumor cells in the bone marrow at 2 weeks. Each dot represents an individual mouse. **P < 0.01 (unpaired t test).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/29/eaat1435/DC1

    Fig. S1. Lymphoma B cells developing in Eμ-myc transgenic mice display extensive intratumor genetic diversity.

    Fig. S2. Frequent chromosome loss in MYC-driven lymphoma B cells.

    Fig. S3. Multicolor fluorescently labeled tumor cells maintain clonal and genomic heterogeneity.

    Fig. S4. Tumor heterogeneity is also detected in the blood.

    Fig. S5. Tumor clonal dominance in nonlymphoid sites is independent of the adaptive immune system.

    Fig. S6. Tumor-specific CD8+ T cells form long-lasting contacts with tumor cells in the bone marrow.

    Fig. S7. Restricted tumor diversity in female recipients as detected by intravital imaging.

    Fig. S8. The immune-mediated restriction of intratumor heterogeneity is not reversible upon in vitro culture.

    Fig. S9. Untransduced lymphoma cells exhibit higher phenotypic diversity in males compared with females.

    Fig. S10. The restriction of tumor heterogeneity requires the adaptive immune system with an important role for CD8+ T cells.

    Fig. S11. Lymphoma cells with an X-only karyotype are less diverse in females compared with males.

    Fig. S12. GFP is not more immunogenic in female than in male mice.

    Fig. S13. Putative neoantigens in Eμ-myc tumors.

    Fig. S14. Modest reduction in tumor load upon checkpoint blockade.

    Fig. S15. Restriction of tumor diversity mediated by checkpoint blockade is associated with tumor immunoediting.

    Movies S1 and S2. Lymphoma subclones establish independent niches in the bone marrow.

    Movie S3. CD8+ T cells infiltrate the bone marrow and interact with lymphoma cells.

    Movie S4. CD8+ T cells form stable contacts with lymphoma cells.

    Movie S5. Tumor-reactive but not control CD8+ T cells establish stable contacts with tumor cells in the bone marrow.

    Movie S6. Visualization of lymphoma cell apoptosis after interactions with tumor-reactive CD8+ T cells.

    Movie S7. Lymphoma cell apoptosis after interactions with tumor-reactive CD8+ T cells but not control T cells.

    Table S1. Raw datasets.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Lymphoma B cells developing in Eμ-myc transgenic mice display extensive intratumor genetic diversity.
    • Fig. S2. Frequent chromosome loss in MYC-driven lymphoma B cells.
    • Fig. S3. Multicolor fluorescently labeled tumor cells maintain clonal and genomic heterogeneity.
    • Fig. S4. Tumor heterogeneity is also detected in the blood.
    • Fig. S5. Tumor clonal dominance in nonlymphoid sites is independent of the adaptive immune system.
    • Fig. S6. Tumor-specific CD8+ T cells form long-lasting contacts with tumor cells in the bone marrow.
    • Fig. S7. Restricted tumor diversity in female recipients as detected by intravital imaging.
    • Fig. S8. The immune-mediated restriction of intratumor heterogeneity is not reversible upon in vitro culture.
    • Fig. S9. Untransduced lymphoma cells exhibit higher phenotypic diversity in males compared with females.
    • Fig. S10. The restriction of tumor heterogeneity requires the adaptive immune system with an important role for CD8+ T cells.
    • Fig. S11. Lymphoma cells with an X-only karyotype are less diverse in females compared with males.
    • Fig. S12. GFP is not more immunogenic in female than in male mice.
    • Fig. S13. Putative neoantigens in Eμ-myc tumors.
    • Fig. S14. Modest reduction in tumor load upon checkpoint blockade.
    • Fig. S15. Restriction of tumor diversity mediated by checkpoint blockade is associated with tumor immunoediting.
    • Legends of movies S1 to S7

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    Other Supplementary Material for this manuscript includes the following:

    • Movies S1 and S2 (.mov format). Lymphoma subclones establish independent niches in the bone marrow.
    • Movie S3 (.mov format). CD8+ T cells infiltrate the bone marrow and interact with lymphoma cells.
    • Movie S4 (.mov format). CD8+ T cells form stable contacts with lymphoma cells.
    • Movie S5 (.avi format). Tumor-reactive but not control CD8+ T cells establish stable contacts with tumor cells in the bone marrow.
    • Movie S6 (.avi format). Visualization of lymphoma cell apoptosis after interactions with tumor-reactive CD8+ T cells.
    • Movie S7 (.avi format). Lymphoma cell apoptosis after interactions with tumor-reactive CD8+ T cells but not control T cells.
    • Table S1. Raw datasets.

    Files in this Data Supplement:

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