Chromosomal instability (CIN), the persistent reshuffling of chromosomes during mitosis, is a hallmark of human cancers that contributes to tumor heterogeneity and has been implicated in driving metastasis and altering responses to therapy. Though multiple mechanisms can produce CIN, lagging chromosomes generated from abnormal merotelic attachments are the major cause of CIN in a variety of cell lines, and are expected to predominate in cancer. Here, we quantify CIN in breast cancer using a tumor microarray, matched primary and metastatic samples, and patient-derived organoids from primary breast cancer. Surprisingly, misaligned chromosomes are more common than lagging chromosomes and represent a major source of CIN in primary and metastatic tumors. This feature of breast cancers is conserved in a majority of breast cancer cell lines. Importantly, though a portion of misaligned chromosomes align before anaphase onset, the fraction that remain represents the largest source of CIN in these cells. Metastatic breast cancers exhibit higher rates of CIN than matched primary cancers, primarily due to increases in misaligned chromosomes. Whether CIN causes immune activation or evasion is controversial. We find that misaligned chromosomes result in immune-activating micronuclei substantially less frequently than lagging and bridge chromosomes and that breast cancers with greater frequencies of lagging chromosomes and chromosome bridges recruit more stromal tumor-infiltrating lymphocytes (sTILs). These data indicate misaligned chromosomes represent a major mechanism of CIN in breast cancer and provide support for differential immunostimulatory effects of specific types of CIN.

<div><p>Chromosomal instability (CIN), the persistent reshuffling of chromosomes during mitosis, is a hallmark of human cancers that contributes to tumor heterogeneity and has been implicated in driving metastasis and altering responses to therapy. Though multiple mechanisms can produce CIN, lagging chromosomes generated from abnormal merotelic attachments are the major cause of CIN in a variety of cell lines, and are expected to predominate in cancer. Here, we quantify CIN in breast cancer using a tumor microarray, matched primary and metastatic samples, and patient-derived organoids from primary breast cancer. Surprisingly, misaligned chromosomes are more common than lagging chromosomes and represent a major source of CIN in primary and metastatic tumors. This feature of breast cancers is conserved in a majority of breast cancer cell lines. Importantly, though a portion of misaligned chromosomes align before anaphase onset, the fraction that remain represents the largest source of CIN in these cells. Metastatic breast cancers exhibit higher rates of CIN than matched primary cancers, primarily due to increases in misaligned chromosomes. Whether CIN causes immune activation or evasion is controversial. We find that misaligned chromosomes result in immune-activating micronuclei substantially less frequently than lagging and bridge chromosomes and that breast cancers with greater frequencies of lagging chromosomes and chromosome bridges recruit more stromal tumor-infiltrating lymphocytes. These data indicate misaligned chromosomes represent a major mechanism of CIN in breast cancer and provide support for differential immunostimulatory effects of specific types of CIN.</p>Significance:<p>We surveyed the single-cell landscape of mitotic defects that generate CIN in primary and metastatic breast cancer and relevant models. Misaligned chromosomes predominate, and are less immunostimulatory than other chromosome segregation errors.</p></div>

<p>Figure S5. Chromosomes that are misaligned at anaphase onset are rapidly incorporated into the main DNA masses during anaphase A. A-B. Timelapse analysis of T47D (A) and Cal51 (B) breast cancer cells with endogenously labeled histone H2B and a-tubulin showing the fate of misaligned chromosomes after anaphase entry. In cells that entered anaphase with misaligned chromosomes, the majority of the misaligned chromosomes reincorporated into the main masses of segregating DNA during anaphase A (left columns). A smaller portion of misaligned chromosomes remained distinct from the segregating DNA masses and therefore visible throughout anaphase A (middle column). A small minority of misaligned chromosomes were detectable for at least 2 minutes during anaphase B (right column). n>25 cells with misaligned chromosomes in 3 biological replicates. C-D timing of anaphase and telophase in T47D (C) and Cal51 (D) cells. n>55 cells in 3 biological replicates.</p>

<p>Lagging chromosome</p>

<p>Normal division</p>