Cell-in-cell phenomena across the tree of life.

Cells in obligately multicellular organisms by definition have aligned fitness interests, minimum conflict, and cannot reproduce independently. However, some cells eat other cells within the same body, sometimes called cell cannibalism. Such cell-in-cell events have not been thoroughly discussed in the framework of major transitions to multicellularity. We performed a systematic screening of 508 articles, from which we chose 115 relevant articles in a search for cell-in-cell events across the tree of life, the age of cell-in-cell-related genes, and whether cell-in-cell events are associated with normal multicellular development or cancer. Cell-in-cell events are found across the tree of life, from some unicellular to many multicellular organisms, including non-neoplastic and neoplastic tissue. Additionally, out of the 38 cell-in-cell-related genes found in the literature, 14 genes were over 2.2 billion years old, i.e., older than the common ancestor of some facultatively multicellular taxa. All of this suggests that cell-in-cell events may have originated before the origins of obligate multicellularity. Thus, our results show that cell-in-cell events exist in obligate multicellular organisms, but are not a defining feature of them. The idea of eradicating cell-in-cell events from obligate multicellular organisms as a way of treating cancer, without considering that cell-in-cell events are also part of normal development, should be abandoned.

Epigenome and early selection determine the tumour-immune evolutionary trajectory of colorectal cancer.

Immune system control is a major hurdle that cancer evolution must circumvent. The relative timing and evolutionary dynamics of subclones that have escaped immune control remain incompletely characterized, and how immune-mediated selection shapes the epigenome has received little attention. Here, we infer the genome- and epigenome-driven evolutionary dynamics of tumour-immune coevolution within primary colorectal cancers (CRCs). We utilise our existing CRC multi-region multi-omic dataset that we supplement with high-resolution spatially-resolved neoantigen sequencing data and highly multiplexed imaging of the tumour microenvironment (TME). Analysis of somatic chromatin accessibility alterations (SCAAs) reveals frequent somatic loss of accessibility at antigen presenting genes, and that SCAAs contribute to silencing of neoantigens. We observe that strong immune escape and exclusion occur at the outset of CRC formation, and that within tumours, including at the microscopic level of individual tumour glands, additional immune escape alterations have negligible consequences for the immunophenotype of cancer cells. Further minor immuno-editing occurs during local invasion and is associated with TME reorganisation, but that evolutionary bottleneck is relatively weak. Collectively, we show that immune evasion in CRC follows a "Big Bang" evolutionary pattern, whereby genetic, epigenetic and TME-driven immune evasion acquired by the time of transformation defines subsequent cancer-immune evolution.

Testing Adaptive Therapy Protocols Using Gemcitabine and Capecitabine in a Preclinical Model of Endocrine-Resistant Breast Cancer.

Adaptive therapy, an ecologically inspired approach to cancer treatment, aims to overcome resistance and reduce toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life instead of killing the maximum number of cancer cells. In preparation for a clinical trial, we used endocrine-resistant MCF7 breast cancer to stimulate second-line therapy and tested adaptive therapy using capecitabine, gemcitabine, or their combination in a mouse xenograft model. Dose modulation adaptive therapy with capecitabine alone increased survival time relative to MTD but not statistically significantly (HR = 0.22, 95% CI = 0.043-1.1, p = 0.065). However, when we alternated the drugs in both dose modulation (HR = 0.11, 95% CI = 0.024-0.55, p = 0.007) and intermittent adaptive therapies, the survival time was significantly increased compared to high-dose combination therapy (HR = 0.07, 95% CI = 0.013-0.42, p = 0.003). Overall, the survival time increased with reduced dose for both single drugs (p < 0.01) and combined drugs (p < 0.001), resulting in tumors with fewer proliferation cells (p = 0.0026) and more apoptotic cells (p = 0.045) compared to high-dose therapy. Adaptive therapy favors slower-growing tumors and shows promise in two-drug alternating regimens instead of being combined.

A Mouse-Specific Model to Detect Genes under Selection in Tumors.

The mouse is a widely used model organism in cancer research. However, no computational methods exist to identify cancer driver genes in mice due to a lack of labeled training data. To address this knowledge gap, we adapted the GUST (Genes Under Selection in Tumors) model, originally trained on human exomes, to mouse exomes via transfer learning. The resulting tool, called GUST-mouse, can estimate long-term and short-term evolutionary selection in mouse tumors, and distinguish between oncogenes, tumor suppressor genes, and passenger genes using high-throughput sequencing data. We applied GUST-mouse to analyze 65 exomes of mouse primary breast cancer models and 17 exomes of mouse leukemia models. Comparing the predictions between cancer types and between human and mouse tumors revealed common and unique driver genes. The GUST-mouse method is available as an open-source R package on github.

Testing Adaptive Therapy Protocols using Gemcitabine and Capecitabine on a Mouse Model of Endocrine-Resistant Breast Cancer.

Highly effective cancer therapies often face limitations due to acquired resistance and toxicity. Adaptive therapy, an ecologically inspired approach, seeks to control therapeutic resistance and minimize toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life over maximum cell kill. In preparation for a clinical trial in breast cancer, we used large populations of MCF7 cells to rapidly generate endocrine-resistance breast cancer cell line. We then mimicked second line therapy in ER+ breast cancers by treating the endocrine-resistant MCF7 cells in a mouse xenograft model to test adaptive therapy with capecitabine, gemcitabine, or the combination of those two drugs. Dose-modulation adaptive therapy with capecitabine alone increased survival time relative to MTD, but not statistically significant (HR: 0.22, 95% CI 0.043- 1.1 P = 0.065). However, when we alternated the drugs in both dose modulation (HR = 0.11, 95% CI: 0.024 - 0.55, P = 0.007) and intermittent adaptive therapies significantly increased survival time compared to high dose combination therapy (HR = 0.07, 95% CI: 0.013 - 0.42; P = 0.003). Overall, survival time increased with reduced dose for both single drugs (P < 0.01) and combined drugs (P < 0.001). Adaptive therapy protocols resulted in tumors with lower proportions of proliferating cells (P = 0.0026) and more apoptotic cells (P = 0.045). The results show that Adaptive therapy outperforms high-dose therapy in controlling endocrine-resistant breast cancer, favoring slower-growing tumors, and showing promise in two-drug alternating regimens.

Cell-in-cell phenomena across the tree of life.

Cells in obligately multicellular organisms by definition have aligned fitness interests, minimum conflict, and cannot reproduce independently. However, some cells eat other cells within the same body, sometimes called cell cannibalism. Such cell-in-cell events have not been thoroughly discussed in the framework of major transitions to multicellularity. We performed a systematic review of 508 articles to search for cell-in-cell events across the tree of life, the age of cell-in-cell-related genes, and whether cell-in-cell events are associated with normal multicellular development or cancer. Out of the 38 cell-in-cell-related genes found in the literature, 14 genes were over 2.2 billion years old, i.e., older than the common ancestor of some facultatively multicellular taxa. Therefore, we propose that cell-in-cell events originated before the origins of obligate multicellularity. Cell-in-cell events are found almost everywhere: across some unicellular and many multicellular organisms, mostly in malignant rather than benign tissue, and in non-neoplastic cells. Thus, our results show that cell-in-cell events exist in obligate multicellular organisms, but are not a defining feature of them. The idea of eradicating cell-in-cell events from obligate multicellular organisms as a way of treating cancer, without considering that cell-in-cell events are also part of normal development, should be abandoned.

Growth Dynamics of Ductal Carcinoma in Situ Recapitulate Normal Breast Development.

Ductal carcinoma in situ (DCIS) and invasive breast cancer share many morphologic, proteomic, and genomic alterations. Yet in contrast to invasive cancer, many DCIS tumors do not progress and may remain indolent over decades. To better understand the heterogenous nature of this disease, we reconstructed the growth dynamics of 18 DCIS tumors based on the geo-spatial distribution of their somatic mutations. The somatic mutation topographies revealed that DCIS is multiclonal and consists of spatially discontinuous subclonal lesions. Here we show that this pattern of spread is consistent with a new 'Comet' model of DCIS tumorigenesis, whereby multiple subclones arise early and nucleate the buds of the growing tumor. The discontinuous, multiclonal growth of the Comet model is analogous to the branching morphogenesis of normal breast development that governs the rapid expansion of the mammary epithelium during puberty. The branching morphogenesis-like dynamics of the proposed Comet model diverges from the canonical model of clonal evolution, and better explains observed genomic spatial data. Importantly, the Comet model allows for the clinically relevant scenario of extensive DCIS spread, without being subjected to the selective pressures of subclone competition that promote the emergence of increasingly invasive phenotypes. As such, the normal cell movement inferred during DCIS growth provides a new explanation for the limited risk of progression in DCIS and adds biologic rationale for ongoing clinical efforts to reduce DCIS overtreatment.

Stronger Together: Cancer Clones Cooperate to Alleviate Growth Barriers in Critical Cancer Progression Transitions.

Hershey and colleagues recently showed how clones in a triple-negative breast cancer cell line cooperate for their mutual fitness benefit. In this system, clones exchange soluble metabolites to increase their in vitro growth rate at low population densities, therefore mitigating the documented growth barrier that reduces individual fitness in small tumor cell populations (Allee effect). Such cooperation could aid important transitions in cancer progression in which cancer cell populations are small, like invasion or metastasis. Using orthotopic transplantation, the authors demonstrate that this cooperation is functional in one such transition in vivo, increasing the metastatic load and number of metastases, which are usually polyclonal. Together, these findings highlight the need to consider ecologic interactions to properly understand tumor growth dynamics, and how they complement the standing evolutionary model of cancer progression in our quest to understand and treat cancer.

The relationship between diet, plasma glucose, and cancer prevalence across vertebrates.

Could diet and mean plasma glucose concentration (MPGluC) explain the variation in cancer prevalence across species? We collected diet, MPGluC, and neoplasia data for 160 vertebrate species from existing databases. We found that MPGluC negatively correlates with cancer and neoplasia prevalence, mostly of gastrointestinal organs. Trophic level positively correlates with cancer and neoplasia prevalence even after controlling for species MPGluC. Most species with high MPGluC (50/78 species = 64.1%) were birds. Most species in high trophic levels (42/53 species = 79.2%) were reptiles and mammals. Our results may be explained by the evolution of insulin resistance in birds which selected for loss or downregulation of genes related to insulin-mediated glucose import in cells. This led to higher MPGluC, intracellular caloric restriction, production of fewer reactive oxygen species and inflammatory cytokines, and longer telomeres contributing to longer longevity and lower neoplasia prevalence in extant birds relative to other vertebrates.

Cancer Prevalence Across Vertebrates.

Cancer is pervasive across multicellular species, but what explains differences in cancer prevalence across species? Using 16,049 necropsy records for 292 species spanning three clades (amphibians, sauropsids and mammals) we found that neoplasia and malignancy prevalence increases with adult weight (contrary to Peto's Paradox) and somatic mutation rate, but decreases with gestation time. Evolution of cancer susceptibility appears to have undergone sudden shifts followed by stabilizing selection. Outliers for neoplasia prevalence include the common porpoise (<1.3%), the Rodrigues fruit bat (<1.6%) the black-footed penguin (<0.4%), ferrets (63%) and opossums (35%). Discovering why some species have particularly high or low levels of cancer may lead to a better understanding of cancer syndromes and novel strategies for the management and prevention of cancer.

Is chimerism associated with cancer across the tree of life?

Chimerism is a widespread phenomenon across the tree of life. It is defined as a multicellular organism composed of cells from other genetically distinct entities. This ability to 'tolerate' non-self cells may be linked to susceptibility to diseases like cancer. Here we test whether chimerism is associated with cancers across obligately multicellular organisms in the tree of life. We classified 12 obligately multicellular taxa from lowest to highest chimerism levels based on the existing literature on the presence of chimerism in these species. We then tested for associations of chimerism with tumour invasiveness, neoplasia (benign or malignant) prevalence and malignancy prevalence in 11 terrestrial mammalian species. We found that taxa with higher levels of chimerism have higher tumour invasiveness, though there was no association between malignancy or neoplasia and chimerism among mammals. This suggests that there may be an important biological relationship between chimerism and susceptibility to tissue invasion by cancerous cells. Studying chimerism might help us identify mechanisms underlying invasive cancers and also could provide insights into the detection and management of emerging transmissible cancers.

Bridging clinic and wildlife care with AI-powered pan-species computational pathology.

Cancers occur across species. Understanding what is consistent and varies across species can provide new insights into cancer initiation and evolution, with significant implications for animal welfare and wildlife conservation. We build a pan-species cancer digital pathology atlas (panspecies.ai) and conduct a pan-species study of computational comparative pathology using a supervised convolutional neural network algorithm trained on human samples. The artificial intelligence algorithm achieves high accuracy in measuring immune response through single-cell classification for two transmissible cancers (canine transmissible venereal tumour, 0.94; Tasmanian devil facial tumour disease, 0.88). In 18 other vertebrate species (mammalia = 11, reptilia = 4, aves = 2, and amphibia = 1), accuracy (range 0.57-0.94) is influenced by cell morphological similarity preserved across different taxonomic groups, tumour sites, and variations in the immune compartment. Furthermore, a spatial immune score based on artificial intelligence and spatial statistics is associated with prognosis in canine melanoma and prostate tumours. A metric, named morphospace overlap, is developed to guide veterinary pathologists towards rational deployment of this technology on new samples. This study provides the foundation and guidelines for transferring artificial intelligence technologies to veterinary pathology based on understanding of morphological conservation, which could vastly accelerate developments in veterinary medicine and comparative oncology.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Life history and cancer in birds: clutch size predicts cancer.

Cancer is a disease that affects nearly all multicellular life, including birds. However, little is known about what factors explain the variance in cancer prevalence among species. Litter size is positively correlated with cancer prevalence in managed species of mammals, and larger body size, but not incubation or nestling period, is linked to tumor prevalence in wild birds. Also, birds that produce more elaborate sexual traits are expected to have fewer resources for cancer defenses and thus higher cancer prevalence. In this study, we examined whether cancer prevalence is associated with a wide variety of life history traits (clutch size, incubation length, body mass, lifespan, and the extent of sexual dimorphism) across 108 species of managed birds in 25 different zoological facilities, sanctuaries, and veterinary clinics. We found that clutch size was positively correlated with cancer and neoplasia (both benign and malignant) prevalence, even after controlling for body mass. Cancer prevalence was not associated with incubation length, body mass, lifespan, or sexual dimorphism. The positive correlations of clutch size with cancer prevalence and neoplasia prevalence suggest that there may be life-history trade-offs between reproductive investment and somatic maintenance (in the form of cancer prevention mechanisms) in managed birds.

Cancer Prevalence Across Vertebrates.

Cancer is pervasive across multicellular species. Are there any patterns that can explain differences in cancer prevalence across species? Using 16,049 necropsy records for 292 species spanning three clades (amphibians, sauropsids and mammals) we found that neoplasia and malignancy prevalence increases with adult weight and decreases with gestation time, contrary to Peto’s Paradox. Evolution of cancer susceptibility appears to have undergone sudden shifts followed by stabilizing selection. Outliers for neoplasia prevalence include the common porpoise (<1.3%), the Rodrigues fruit bat (<1.6%) the black-footed penguin (<0.4%), ferrets (63%) and opossums (35%). Discovering why some species have particularly high or low levels of cancer may lead to a better understanding of cancer syndromes and novel strategies for the management and prevention of cancer.

Elephant TP53-RETROGENE 9 induces transcription-independent apoptosis at the mitochondria.

Approximately 20 TP53 retrogenes exist in the African and Asian elephant genomes (Loxodonta Africana, Elephas Maximus) in addition to a conserved TP53 gene that encodes a full-length protein. Elephant TP53-RETROGENE 9 (TP53-R9) encodes a p53 protein (p53-R9) that is truncated in the middle of the canonical DNA binding domain. This C-terminally truncated p53 retrogene protein lacks the nuclear localization signals and oligomerization domain of its full-length counterpart. When expressed in human osteosarcoma cells (U2OS), p53-R9 binds to Tid1, the chaperone protein responsible for mitochondrial translocation of human p53 in response to cellular stress. Tid1 expression is required for p53-R9-induced apoptosis. At the mitochondria, p53-R9 binds to the pro-apoptotic BCL-2 family member Bax, which leads to caspase activation, cytochrome c release, and cell death. Our data show, for the first time, that expression of this truncated elephant p53 retrogene protein induces apoptosis in human cancer cells. Understanding the molecular mechanism by which the additional elephant TP53 retrogenes function may provide evolutionary insight that can be utilized for the development of therapeutics to treat human cancers.

Diet, Microbes, and Cancer Across the Tree of Life: a Systematic Review.

PURPOSE OF REVIEW: Cancers are a leading cause of death in humans and for many other species. Diet has often been associated with cancers, and the microbiome is an essential mediator between diet and cancers. Here, we review the work on cancer and the microbiome across species to search for broad patterns of susceptibility associated with different microbial species. RECENT FINDINGS: Some microbes, such as Helicobacter bacteria, papillomaviruses, and the carnivore-associated Fusobacteria, consistently induce tumorigenesis in humans and other species. Other microbes, such as the milk-associated Lactobacillus, consistently inhibit tumorigenesis in humans and other species. We systematically reviewed over a thousand published articles and identified links between diet, microbes, and cancers in several species of mammals, birds, and flies. Future work should examine a larger variety of host species to discover new model organisms for human preclinical trials, to better understand the observed variance in cancer prevalence across species, and to discover which microbes and diets are associated with cancers across species. Ultimately, this could help identify microbial and dietary interventions to diagnose, prevent, and treat cancers in humans as well as other animals.

In Silico Investigations of Multi-Drug Adaptive Therapy Protocols.

The standard of care for cancer patients aims to eradicate the tumor by killing the maximum number of cancer cells using the maximum tolerated dose (MTD) of a drug. MTD causes significant toxicity and selects for resistant cells, eventually making the tumor refractory to treatment. Adaptive therapy aims to maximize time to progression (TTP), by maintaining sensitive cells to compete with resistant cells. We explored both dose modulation (DM) protocols and fixed dose (FD) interspersed with drug holiday protocols. In contrast to previous single drug protocols, we explored the determinants of success of two-drug adaptive therapy protocols, using an agent-based model. In almost all cases, DM protocols (but not FD protocols) increased TTP relative to MTD. DM protocols worked well when there was more competition, with a higher cost of resistance, greater cell turnover, and when crowded proliferating cells could replace their neighbors. The amount that the drug dose was changed, mattered less. The more sensitive the protocol was to tumor burden changes, the better. In general, protocols that used as little drug as possible, worked best. Preclinical experiments should test these predictions, especially dose modulation protocols, with the goal of generating successful clinical trials for greater cancer control.

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Somatic whole genome dynamics of precancer in Barrett's esophagus reveals features associated with disease progression.

While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett's esophagus compared to 40 Barrett's patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett's tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett's versus those who progress to cancer is acquisition and expansion of TP53-/- cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett's esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies.