Microfluidics guided by deep learning for cancer immunotherapy screening.

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.

Adaptive phase I-II clinical trial designs identifying optimal biological doses for targeted agents and immunotherapies.

Targeted agents and immunotherapies have revolutionized cancer treatment, offering promising options for various cancer types. Unlike traditional therapies the principle of "more is better" is not always applicable to these new therapies due to their unique biomedical mechanisms. As a result, various phase I-II clinical trial designs have been proposed to identify the optimal biological dose that maximizes the therapeutic effect of targeted therapies and immunotherapies by jointly monitoring both efficacy and toxicity outcomes. This review article examines several innovative phase I-II clinical trial designs that utilize accumulated efficacy and toxicity outcomes to adaptively determine doses for subsequent patients and identify the optimal biological dose, maximizing the overall therapeutic effect. Specifically, we highlight three categories of phase I-II designs: efficacy-driven, utility-based, and designs incorporating multiple efficacy endpoints. For each design, we review the dose-outcome model, the definition of the optimal biological dose, the dose-finding algorithm, and the software for trial implementation. To illustrate the concepts, we also present two real phase I-II trial examples utilizing the EffTox and ISO designs. Finally, we provide a classification tree to summarize the designs discussed in this article.

SSMD: a semi-supervised approach for a robust cell type identification and deconvolution of mouse transcriptomics data.

Deconvolution of mouse transcriptomic data is challenged by the fact that mouse models carry various genetic and physiological perturbations, making it questionable to assume fixed cell types and cell type marker genes for different data set scenarios. We developed a Semi-Supervised Mouse data Deconvolution (SSMD) method to study the mouse tissue microenvironment. SSMD is featured by (i) a novel nonparametric method to discover data set-specific cell type signature genes; (ii) a community detection approach for fixing cell types and their marker genes; (iii) a constrained matrix decomposition method to solve cell type relative proportions that is robust to diverse experimental platforms. In summary, SSMD addressed several key challenges in the deconvolution of mouse tissue data, including: (i) varied cell types and marker genes caused by highly divergent genotypic and phenotypic conditions of mouse experiment; (ii) diverse experimental platforms of mouse transcriptomics data; (iii) small sample size and limited training data source and (iv) capable to estimate the proportion of 35 cell types in blood, inflammatory, central nervous or hematopoietic systems. In silico and experimental validation of SSMD demonstrated its high sensitivity and accuracy in identifying (sub) cell types and predicting cell proportions comparing with state-of-the-arts methods. A user-friendly R package and a web server of SSMD are released via https://github.com/xiaoyulu95/SSMD.

PLUS: Predicting cancer metastasis potential based on positive and unlabeled learning.

Metastatic cancer accounts for over 90% of all cancer deaths, and evaluations of metastasis potential are vital for minimizing the metastasis-associated mortality and achieving optimal clinical decision-making. Computational assessment of metastasis potential based on large-scale transcriptomic cancer data is challenging because metastasis events are not always clinically detectable. The under-diagnosis of metastasis events results in biased classification labels, and classification tools using biased labels may lead to inaccurate estimations of metastasis potential. This issue is further complicated by the unknown metastasis prevalence at the population level, the small number of confirmed metastasis cases, and the high dimensionality of the candidate molecular features. Our proposed algorithm, called Positive and unlabeled Learning from Unbalanced cases and Sparse structures (PLUS), is the first to use a positive and unlabeled learning framework to account for the under-detection of metastasis events in building a classifier. PLUS is specifically tailored for studying metastasis that deals with the unbalanced instance allocation as well as unknown metastasis prevalence, which are not considered by other methods. PLUS achieves superior performance on synthetic datasets compared with other state-of-the-art methods. Application of PLUS to The Cancer Genome Atlas Pan-Cancer gene expression data generated metastasis potential predictions that show good agreement with the clinical follow-up data, in addition to predictive genes that have been validated by independent single-cell RNA-sequencing datasets.

Loss-of-Function Genetic Screening Identifies Aldolase A as an Essential Driver for Liver Cancer Cell Growth Under Hypoxia.

BACKGROUND AND AIMS: Hypoxia is a common feature of the tumor microenvironment (TME), which promotes tumor progression, metastasis, and therapeutic drug resistance through a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of HCC. APPROACH AND RESULTS: From a genome-wide CRISPR/CRISPR-associated 9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion and consequently inhibits tumor growth. Supplementation with lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by hypoxia-inducible factor-1α and fat mass and obesity-associated protein-mediated N6 -methyladenosine modification through transcriptional and posttranscriptional regulation, respectively. Analysis of The Cancer Genome Atlas shows that elevated levels of ALDOA are significantly correlated with poor prognosis of patients with HCC. In a screen of Food and Drug Administration-approved drugs based on structured hierarchical virtual platforms, we identified the sulfamonomethoxine derivative compound 5 (cpd-5) as a potential inhibitor to target ALDOA, evidenced by the antitumor activity of cpd-5 in preclinical patient-derived xenograft models of HCC. CONCLUSIONS: Our work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in the hypoxic TME is a promising therapeutic strategy for treating HCC.

RNA-binding protein RALY reprogrammes mitochondrial metabolism via mediating miRNA processing in colorectal cancer.

OBJECTIVE: Dysregulated cellular metabolism is a distinct hallmark of human colorectal cancer (CRC). However, metabolic programme rewiring during tumour progression has yet to be fully understood. DESIGN: We analysed altered gene signatures during colorectal tumour progression, and used a complex of molecular and metabolic assays to study the regulation of metabolism in CRC cell lines, human patient-derived xenograft mouse models and tumour organoid models. RESULTS: We identified a novel RNA-binding protein, RALY (also known as hnRNPCL2), that is highly associated with colorectal tumour aggressiveness. RALY acts as a key regulatory component in the Drosha complex, and promotes the post-transcriptional processing of a specific subset of miRNAs (miR-483, miR-676 and miR-877). These miRNAs systematically downregulate the expression of the metabolism-associated genes (ATP5I, ATP5G1, ATP5G3 and CYC1) and thereby reprogramme mitochondrial metabolism in the cancer cell. Analysis of The Cancer Genome Atlas (TCGA) reveals that increased levels of RALY are associated with poor prognosis in the patients with CRC expressing low levels of mitochondrion-associated genes. Mechanistically, induced processing of these miRNAs is facilitated by their N6-methyladenosine switch under reactive oxygen species (ROS) stress. Inhibition of the m6A methylation abolishes the RALY recognition of the terminal loop of the pri-miRNAs. Knockdown of RALY inhibits colorectal tumour growth and progression in vivo and in organoid models. CONCLUSIONS: Collectively, our results reveal a critical metabolism-centric role of RALY in tumour progression, which may lead to cancer therapeutics targeting RALY for treating CRC.

Metabolic interventions: A new insight into the cancer immunotherapy.

Metabolic reprogramming confers cancer cells plasticity and viability under harsh conditions. Such active alterations lead to cell metabolic dependency, which can be exploited as an attractive target in development of effective antitumor therapies. Similar to cancer cells, activated T cells also execute global metabolic reprogramming for their proliferation and effector functions when recruited to the tumor microenvironment (TME). However, the high metabolic activity of rapidly proliferating cancer cells can compete for nutrients with immune cells in the TME, and consequently, suppressing their anti-tumor functions. Thus, therapeutic strategies could aim to restore T cell metabolism and anti-tumor responses in the TME by targeting the metabolic dependence of cancer cells. In this review, we highlight current research progress on metabolic reprogramming and the interplay between cancer cells and immune cells. We also discuss potential therapeutic intervention strategies for targeting metabolic pathways to improve cancer immunotherapy efficacy.

TP53 loss creates therapeutic vulnerability in colorectal cancer.

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Oncogenic KRAS Reduces Expression of FGF21 in Acinar Cells to Promote Pancreatic Tumorigenesis in Mice on a High-Fat Diet.

BACKGROUND & AIMS: Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis. METHODS: We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (KrasG12D/+ mice) and fElasCreERT mice (controls). KrasG12D/+ mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate. RESULTS: Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed KrasG12D/+ had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from KrasG12D/+ mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed KrasG12D/+ mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed KrasG12D/+ mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed KrasG12D/+ mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle. CONCLUSIONS: Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer.

Profiling molecular regulators of recurrence in chemorefractory triple-negative breast cancers.

BACKGROUND: Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development. METHODS: We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes. RESULTS: Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2. CONCLUSIONS: We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation.

The ATM kinase induces microRNA biogenesis in the DNA damage response.

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis.

Acoustic assembly of cell spheroids in disposable capillaries.

Multicellular spheroids represent a promising approach to mimic 3D tissues in vivo for emerging applications in regenerative medicine, therapeutic screening, and drug discovery. Conventional spheroid fabrication methods, such as the hanging drop method, suffer from low-throughput, long time, complicated procedure, and high heterogeneity in spheroid size. In this work, we report a simple yet reliable acoustic method to rapidly assemble cell spheroids in capillaries in a replicable and scalable manner. Briefly, by introducing a coupled standing surface acoustic wave, we are able to generate a linear pressure node array with 300 trapping nodes simultaneously. This enables us to continuously fabricate spheroids in a high-throughput manner with minimal variability in spheroid size. In a proof of concept application, we fabricated cell spheroids of mouse embryonic carcinoma (P19) cells, which grew well and retained differentiation potential in vitro. Based on the advantages of the non-invasive, contactless and label-free acoustic cell manipulation, our method employs the coupling strategy to assemble cells in capillaries, and further advances 3D spheroid assembly technology in an easy, cost-efficient, consistent, and high-throughput manner. This method could further be adapted into a novel 3D biofabrication approach to replicate compilated tissues and organs for a wide set of biomedical applications.

A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation.

A prompt and efficient DNA damage response (DDR) eliminates the detrimental effects of DNA lesions in eukaryotic cells. Basic and preclinical studies suggest that the DDR is one of the primary anti-cancer barriers during tumorigenesis. The DDR involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play an important role. In the current study, we identified a novel lncRNA, lncRNA-JADE, that is induced after DNA damage in an ataxia-telangiectasia mutated (ATM)-dependent manner. LncRNA-JADE transcriptionally activates Jade1, a key component in the HBO1 (human acetylase binding to ORC1) histone acetylation complex. Consequently, lncRNA-JADE induces histone H4 acetylation in the DDR. Markedly higher levels of lncRNA-JADE were observed in human breast tumours in comparison with normal breast tissues. Knockdown of lncRNA-JADE significantly inhibited breast tumour growth in vivo. On the basis of these results, we propose that lncRNA-JADE is a key functional link that connects the DDR to histone H4 acetylation, and that dysregulation of lncRNA-JADE may contribute to breast tumorigenesis.

Prim-O-glucosylcimifugin induces cell cycle arrest and apoptosis in acute lymphoblastic leukemia cells.

Prim-O-glucosylcimifugin is a major constituent in Radix Saposhnikovia that has been long used for the treatment of pyrexia, rheumatism, and cancer in traditional Chinese medicine. However, the molecular and cellular mechanisms remain unknown regarding the therapeutic effect of prim-O-lucosylcimifugin. Here, we investigated the effects of prim-O-glucosylcimifugin on cell cycle progression and apoptosis in human acute lymphoblastic leukemia cells. Prim-O-glucosylcimifugin treatment resulted in marked increases in cell apoptosis and cell cycle arrest at the G2/M phase. Mechanistically, prim-O-glucosylcimifugin induced the degradation of ß-tubulin and downregulated phosphorylated CDK1 levels, a molecular indicator in the G2/M cell cycle arrest. Furthermore, activation of caspase-3, caspase-8, and caspase-9 was involved in the prim-O-glucosylcimifugin-induced apoptosis. Our study reveals the anticancer activity of prim-O-glucosylcimifugin and the potential underlying mechanisms.

Targeting tumor suppressor genes for cancer therapy.

Cancer drugs are broadly classified into two categories: cytotoxic chemotherapies and targeted therapies that specifically modulate the activity of one or more proteins involved in cancer. Major advances have been achieved in targeted cancer therapies in the past few decades, which is ascribed to the increasing understanding of molecular mechanisms for cancer initiation and progression. Consequently, monoclonal antibodies and small molecules have been developed to interfere with a specific molecular oncogenic target. Targeting gain-of-function mutations, in general, has been productive. However, it has been a major challenge to use standard pharmacologic approaches to target loss-of-function mutations of tumor suppressor genes. Novel approaches, including synthetic lethality and collateral vulnerability screens, are now being developed to target gene defects in p53, PTEN, and BRCA1/2. Here, we review and summarize the recent findings in cancer genomics, drug development, and molecular cancer biology, which show promise in targeting tumor suppressors in cancer therapeutics.

Continuous On-Chip Cell Separation Based on Conductivity-Induced Dielectrophoresis with 3D Self-Assembled Ionic Liquid Electrodes.

Dielectrophoresis (DEP) has been widely explored to separate cells for various applications. However, existing DEP devices are limited by the high cost associated with the use of noble metal electrodes, the need of high-voltage electric field, and/or discontinuous separation (particularly for devices without metal electrodes). We developed a DEP device with liquid electrodes, which can be used to continuously separate different types of cells or particles based on positive DEP. The device is made of polydimethylsiloxane (PDMS), and ionic liquid is used to form the liquid electrodes, which has the advantages of low cost and easy fabrication. Moreover, the conductivity gradient is utilized to achieve the DEP-based on-chip cell separation. The device was used to separate polystyrene microbeads and PC-3 human prostate cancer cells with 94.7 and 1.2% of the cells and microbeads being deflected, respectively. This device is also capable of separating live and dead PC-3 cancer cells with 89.8 and 13.2% of the live and dead cells being deflected, respectively. Moreover, MDA-MB-231 human breast cancer cells could be separated from human adipose-derived stem cells (ADSCs) using this device with high purity (81.8 and 82.5% for the ADSCs and MDA-MB-231 cells, respectively). Our data suggest the great potential of cell separation based on conductivity-induced DEP using affordable microfluidic devices with easy operation.

Regulators in the DNA damage response.

Maintenance of genome integrity is essential for the proper function of all cells and organisms. In response to both endogenous and exogenous DNA damaging agents, mammalian cells have evolved a delicate system to sense DNA damage, stop cell cycle progression, modulate cell metabolism, repair damaged DNA, and induce programmed cell death if the damage is too severe. This coordinated global signaling network, namely the DNA damage response (DDR), ensures the genome stability under DNA damaging stress. A variety of regulators have been shown to modulate the activity and levels of key proteins in the DDR, including kinases, phosphatases, ubiquitin ligases, deubiquitinases, and other protein modifying enzymes. Epigenetic regulators, particularly microRNAs and long noncoding RNAs, have been emerging as an important payer of regulation in addition to canonical DNA damage signaling proteins. In this review, we will discuss the functional interaction between the regulators and their targets in the DDR.

Enhanced Microwave Hyperthermia of Cancer Cells with Fullerene.

Hyperthermia generated with various energy sources including microwave has been widely studied for cancer treatment. However, the potential damage due to nontargeted heating of normal tissue is a major hurdle to its widespread application. Fullerene is a potential agent for improving cancer therapy with microwave hyperthermia but is limited by its poor solubility in water for biomedical applications. Here we report a combination therapy for enhanced cancer cell destruction by combining microwave heating with C60-PCNPs consisting of fullerene (C60) encapsulated in Pluronic F127-chitosan nanoparticles (PCNPs) with high water solubility. A cell culture dish integrated with an antenna was fabricated to generate microwave (2.7 GHz) for heating PC-3 human prostate cancer cells either with or without the C60-PCNPs. The cell viability data show that the C60-PCNPs alone have minimal cytotoxicity. The combination of microwave heating and C60-PCNPs is significantly more effective than the microwave heating alone in killing the cancer cells (7.5 versus 42.2% cell survival). Moreover, the combination of microwave heating and C60-PCNPs is significantly more destructive to the cancer cells than the combination of simple water-bath heating (with a similar thermal history to microwave heating) and C60-PCNPs (7.5 versus 32.5% survival) because the C60 in the many nanoparticles taken up by the cells can absorb the microwave energy and convert it into heat to enhance heating inside the cells under microwave irradiation. These data suggest the great potential of targeted heating via fullerene for enhanced cancer treatment by microwave hyperthermia.

Nanodrug-Mediated Thermotherapy of Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) are rare subpopulations of cancer cells that are resistant to conventional chemotherapy and radiotherapy and contribute to cancer metastases and tumor recurrence. Therefore, it is of significance to develop an effective therapy to eliminate the CSCs. Cancer thermotherapy realized by depositing heat into tumor in a minimally invasive way is a promising alternative to the conventional therapies for cancer treatment. However, this method is limited by its inability to target CSCs, potentially allowing the CSCs to survive and re-initiate tumor growth. More recently, nanodrug-mediated thermotherapy has been explored to selectively eliminate CSCs and specifically deposit heat in tumor to spare healthy tissue. Here, we provide a brief overview of the targeting moieties and nanoplatforms used in developing nanodrug-mediated thermotherapy of cancer with particular emphasis on the CSCs, as well as the challenges and potential directions for future research in this emerging field.