Abstract
The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours ...including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.
Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer ...cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.
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•mTRAIL-R promotes KRAS-driven lung and pancreatic cancer growth and metastasis•Human TRAIL-R2 promotes tumor growth, migration, invasion, and metastasis•Endogenous mTRAIL-R constitutively activates Rac1 in vivo in tumors•TRAIL-R2 expression positively correlates with the onset of metastasis in patients
von Karstedt et al. show that mouse TRAIL-R and human TRAIL-R2, but not TRAIL-R1, are important for the progression, invasion, and metastasis of KRAS-mutant tumors through the regulation of Rac-1.
How cancer cells adapt to stress
Bacteria adapt to harsh conditions such as antibiotic exposure by acquiring new mutations, a process called stress-induced mutagenesis. Cipponi
et al.
investigated ...whether similar programs of mutagenesis play a role in the response of cancer cells to targeted therapies. Using in vitro models of intense drug selection and genome-wide functional screens, the authors found evidence for an analogous process in cancer and showed that it is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. This pathway appears to mediate a stress-related switch to error-prone DNA repair, resulting in the generation of mutations that facilitate the emergence of drug resistance.
Science
, this issue p.
1127
An evolutionarily conserved program of adaptive mutagenesis accelerates drug resistance in human cancers.
In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.
Obesity is caused by an imbalance between food intake and energy expenditure (EE). Here we identify a conserved pathway that links signalling through peripheral Y1 receptors (Y1R) to the control of ...EE. Selective antagonism of peripheral Y1R, via the non-brain penetrable antagonist BIBO3304, leads to a significant reduction in body weight gain due to enhanced EE thereby reducing fat mass. Specifically thermogenesis in brown adipose tissue (BAT) due to elevated UCP1 is enhanced accompanied by extensive browning of white adipose tissue both in mice and humans. Importantly, selective ablation of Y1R from adipocytes protects against diet-induced obesity. Furthermore, peripheral specific Y1R antagonism also improves glucose homeostasis mainly driven by dynamic changes in Akt activity in BAT. Together, these data suggest that selective peripheral only Y1R antagonism via BIBO3304, or a functional analogue, could be developed as a safer and more effective treatment option to mitigate diet-induced obesity.
Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. ...Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of
cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events
We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the single-cell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.
Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we ...used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation.
Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral ...T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.
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•Thwarting LEC autophagy prevents lymphocyte egress from the lymph node (LN)•LEC autophagy governs the S1P gradient and the intranodal dynamics of T and NK cells•Loss of LEC autophagy remodels niche-specific LEC phenotypes in tumor-draining LNs•LEC-autophagy blockade blunts efficacy of ICBs by sequestering T and NK cells in the LNs
Houbaert et al. find that defective autophagy in lymphatic endothelial cells (LECs) prevents lymphocyte’s egress from lymph nodes. In tumor-bearing mice, LEC autophagy endorses the recruitment of peripheral T and NK cells to the tumor and ICB-driven antitumor immunity, further underscoring the lymph nodes as critical players in immunotherapy responses.
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway ...within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.
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•Hypoxia presents a moving pocket of resistance in pancreatic ductal adenocarcinoma.•Dual intravital imaging allows live tracking of drug response in hypoxic regions.•Combination with a hypoxia-activated pro-drug alleviates resistance.•This has implications for combatting resistance in a broad range of therapies.
Intravital imaging facilitates the real-time tracking and targeting of moving hypoxic regions within pancreatic ductal adenocarcinoma. Using this approach, Conway et al. alleviate hypoxia-induced resistance to a dual mTORC1/2 inhibitor AZD2014, improving PI3K pathway inhibition and demonstrating a powerful dual imaging modality applicable to targeting other pathways and cancers.
We previously used a pulse-based
assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here we advanced this model ...system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single cell fates both
and
we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.
The oncogenic receptor tyrosine kinase (RTK) ERBB2 is known to dimerize with other EGFR family members, particularly ERBB3, through which it potently activates PI3K signalling. Antibody-mediated ...inhibition of this ERBB2/ERBB3/PI3K axis has been a cornerstone of treatment for ERBB2-amplified breast cancer patients for two decades. However, the lack of response and the rapid onset of relapse in many patients now question the assumption that the ERBB2/ERBB3 heterodimer is the sole relevant effector target of these therapies.
Through a systematic protein-protein interaction screen, we have identified and validated alternative RTKs that interact with ERBB2. Using quantitative readouts of signalling pathway activation and cell proliferation, we have examined their influence upon the mechanism of trastuzumab- and pertuzumab-mediated inhibition of cell growth in ERBB2-amplified breast cancer cell lines and a patient-derived xenograft model.
We now demonstrate that inactivation of ERBB3/PI3K by these therapeutic antibodies is insufficient to inhibit the growth of ERBB2-amplified breast cancer cells. Instead, we show extensive promiscuity between ERBB2 and an array of RTKs from outside of the EGFR family. Paradoxically, pertuzumab also acts as an artificial ligand to promote ERBB2 activation and ERK signalling, through allosteric activation by a subset of these non-canonical RTKs. However, this unexpected activation mechanism also increases the sensitivity of the receptor network to the ERBB2 kinase inhibitor lapatinib, which in combination with pertuzumab, displays a synergistic effect in single-agent resistant cell lines and PDX models.
The interaction of ERBB2 with a number of non-canonical RTKs activates a compensatory signalling response following treatment with pertuzumab, although a counter-intuitive combination of ERBB2 antibody therapy and a kinase inhibitor can overcome this innate therapeutic resistance.
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