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Purpose Chimeric antigen receptor (CAR) T cell cancer immunotherapies have shown remarkable results in patients with hematological malignancies and represent the first approved genetically modified cellular therapies. However, not all blood cancer patients respond favorably, serious side effects have been reported, and the treatment of solid tumors has been a challenge. An imaging tool for visualizing the variety of CAR-T cell products in use and being explored could provide important patient-specific data on CAR-T cell location to inform on potential success or failure of treatment as well as off-target toxicities. Fluorine-19 (19F) magnetic resonance imaging (MRI) allows for the noninvasive detection of 19F perfluorocarbon (PFC) labeled cells. Our objective was to visualize PFC-labeled (PFC +) CAR-T cells in a mouse model of leukemia using clinical field strength (3 Tesla) 19F MRI and compare the cytotoxicity of PFC + versus unlabeled CAR-T cells. Procedures NSG mice (n = 17) received subcutaneous injections of CD19 + human B cell leukemia cells (NALM6) expressing firefly luciferase in their left hind flank (1 × 106). Twenty-one days later, each mouse received an intratumoral injection of 10 × 106 PFC + CD19-targeted CAR-T cells (n = 6), unlabeled CD19-targeted CAR-T cells (n = 3), PFC + untransduced T cells (n = 5), or an equivalent volume of saline (n = 3). 19F MRI was performed on mice treated with PFC + CAR-T cells days 1, 3, and 7 post-treatment. Bioluminescence imaging (BLI) was performed on all mice days − 1, 5, 10, and 14 post-treatment to monitor tumor response. Results PFC + CAR-T cells were successfully detected in tumors using 19F MRI on days 1, 3, and 7 post-injection. In vivo BLI data revealed that mice treated with PFC + or PFC − CAR-T cells had significantly lower tumor burden by day 14 compared to untreated mice and mice treated with PFC + untransduced T cells (p Conclusions Our studies demonstrate that clinical field strength 19F MRI can be used to visualize PFC + CAR-T cells for up to 7 days post–intratumoral injection. Importantly, PFC labeling did not significantly affect in vivo CAR-T cell cytotoxicity. These imaging tools may have broad applications for tracking emerging CAR-T cell therapies in preclinical models and may eventually be useful for the detection of CAR-T cells in patients where localized injection of CAR-T cells is being pursued.

There is momentum towards implementing patient-derived xenograft models (PDX) in cancer research to reflect the histopathology, tumour behavior, and metastatic properties observed in the original tumour. These models are more predictive of clinical outcomes and are superior to cell lines for preclinical drug evaluation and therapeutic strategies. To study PDX cells preclinically, we used both bioluminescence imaging (BLI) to evaluate cell viability and magnetic particle imaging (MPI), an emerging imaging technology to allow for detection and quantification of iron nanoparticles. The goal of this study was to develop the first successful iron labeling method of breast cancer cells derived from patient brain metastases and validate this method with imaging during tumour development.Luciferase expressing human breast cancer PDX cells (F2-7) were successfully labeled after incubation with micron-sized iron oxide particles (MPIO; 25 μg Fe/mL). NOD/SCID/ILIIrg-/- (n=5) mice received injections of 1×106 iron-labeled F2-7 cells into the fourth mammary fat pad (MFP). BLI was performed longitudinally to day 49 and MPI was performed up to day 28. In vivo BLI revealed that signal increased over time with tumour development. MPI revealed decreasing signal in the tumours and increasing signal in the liver region over time.Here, we demonstrate the first application of MPI to monitor the growth of a PDX MFP tumour. To accomplish this, we also demonstrate the first successful labeling of PDX cells with iron oxide particles. Imaging of PDX cells provides a powerful system to better develop personalized therapies targeting breast cancer brain metastasis.

Cell-based cancer immunotherapies are becoming a routine part of the armamentarium against cancer. While remarkable successes have been seen, including durable remissions, not all patients will benefit from these therapies and many can suffer from life-threatening side effects. These differences in efficacy and safety across patients and across tumor types (e.g., blood vs. solid), are thought to be due to differences in how well the immune cells traffic to their target tissue (e.g., tumor, lymph nodes, etc.) whilst avoiding non-target tissues. Across patient variability can also stem from whether the cells interact with (i.e., communicate with) their intended target cells (e.g., cancer cells), as well as if they proliferate and survive long enough to yield potent and long-lasting therapeutic effects. However, many cell-based therapies are monitored by relatively simple blood tests that lack any spatial information and do not reflect how many immune cells have ended up at particular tissues. The ex vivo labeling and imaging of infused therapeutic immune cells can provide a more precise and dynamic understanding of whole-body immune cell biodistribution, expansion, viability, and activation status in individual patients. In recent years numerous cellular imaging technologies have been developed that may provide this much-needed information on immune cell fate. For this review, we summarize various ex vivo labeling and imaging approaches that allow for tracking of cellular immunotherapies for cancer. Our focus is on clinical imaging modalities and summarize the progression from experimental to therapeutic settings. The imaging information provided by these technologies can potentially be used for many purposes including improved real-time understanding of therapeutic efficacy and potential side effects in individual patients after cell infusion; the ability to more readily compare new therapeutic cell designs to current designs for various parameters such as improved trafficking to target tissues and avoidance of non-target tissues; and the long-term ability to identify patient populations that are likely to be positive responders and at low-risk of side effects.

New ways to target and treat metastatic disease are urgently needed. Tumor "self-homing" describes the recruitment of circulating tumor cells (CTCs) back to a previously excised primary tumor location, contributing to tumor recurrence, as well as their migration to established metastatic lesions. Recently, self-homing CTCs have been exploited as delivery vehicles for anti-cancer therapeutics in preclinical primary tumor models. However, the ability of CTCs to self-home and treat metastatic disease is largely unknown. Methods: Here, we used bioluminescence imaging (BLI) to explore whether systemically administered CTCs home to metastatic lesions and if CTCs armed with both a reporter gene and a cytotoxic prodrug gene therapy can be used to visualize and treat metastatic disease. Results: BLI performed over time revealed a remarkable ability of CTCs to home to and treat tumors throughout the body. Excitingly, metastatic tumor burden in mice that received therapeutic CTCs was lower compared to mice receiving control CTCs. Conclusion: This study demonstrates the noteworthy ability of experimental CTCs to home to disseminated breast cancer lesions. Moreover, by incorporating a prodrug gene therapy system into our self-homing CTCs, we show exciting progress towards effective and targeted delivery of gene-based therapeutics to treat both primary and metastatic lesions.

Purpose: New ways to target and treat metastatic disease are urgently needed. Tumor “self-homing” describes the recruitment of circulating tumor cells (CTCs) back to a previously excised primary tumor location, contributing to tumor recurrence, as well as their migration to established metastatic lesions. Recently, self-homing CTCs have been exploited as delivery vehicles for anti-cancer therapeutics in preclinical primary tumor models. However, the ability of CTCs to self-home and treat metastatic disease is largely unknown. Methods: Here, we employ molecular imaging to explore whether systemically-administered CTCs home to metastatic lesions and if CTCs armed with both a reporter gene and a cytotoxic prodrug gene therapy can be used to visualize and treat metastatic disease. Results: Bioluminescence imaging (BLI) performed over time revealed a remarkable ability of CTCs to home to primary and metastatic tumors throughout the body. Mice that received therapeutic CTCs had less BLI signal as well as less primary tumour burden than control mice. Preliminary data also showed self-homing therapeutic CTCs may be effective at treating disseminated breast cancer metastases. Conclusion: Using dual-luciferase BLI, this study demonstrates the noteworthy ability of experimental CTCs to home to disseminated breast cancer lesions. Moreover, by incorporating a prodrug gene therapy system into our self-homing CTCs, we show exciting progress towards effective and targeted delivery of gene-based therapeutics to treat both primary and metastatic lesions.

Noninvasive molecular-genetic imaging of cells expressing imaging reporter genes is an invaluable approach for longitudinal monitoring of the biodistribution and viability of cancer cells and cell-based therapies in preclinical models and patients. However, labeling cells with reporter genes often relies on using gene transfer methods that randomly integrate the reporter genes into the genome, which may cause unwanted and serious detrimental effects. To overcome this, we have developed CRISPR-Cas9 tools to edit cells at the adeno-associated virus site 1 (AAVS1) safe harbour with a large donor construct (∼6.3 kilobases) encoding an antibiotic resistance gene and reporter genes for bioluminescence (BLI) and fluorescence imaging. HEK293T cells were transfected with a dual plasmid system encoding the Cas9 endonuclease and an AAVS1-targeted guide RNA in one plasmid, and a donor plasmid encoding a puromycin resistance gene, tdTomato and firefly luciferase flanked by AAVS1 homology arms. Puromycin-resistant clonal cells were isolated and AAVS1 integration was confirmed via PCR and sequencing of the PCR product.

Purpose. The combined use of anatomical magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging (BLI) allows for sensitive and improved monitoring of brain metastasis in preclinical cancer models. By using these complementary technologies, we can acquire measurements of viable single cell arrest in the brain after systemic administration, the clearance and/or retention of these cells thereafter, the growth into overt tumours, and quantification of tumour volume and relative cancer cell viability over time. While BLI is very useful in measuring cell viability, some considerations have been reported using cells engineered with luciferase such as increased tumour volume variation, changes in pattern of metastatic disease, and inhibition of in vivo tumour growth. Procedures. Here, we apply cellular and anatomical MRI to evaluate in vivo growth differences between iron oxide labeled naïve (4T1BR5) and luciferase-expressing (4T1BR5-FLuc-GFP) murine brain-seeking breast cancer cells. Balb/C mice received an intracardiac injection of 20,000 cells and were imaged with MRI on days 0 and 14. Mice that received 4T1BR5-FLuc-GFP cells were also imaged with BLI on days 0 and 14. Results. The number of signal voids in the brain (representing iron-labeled cancer cells) on day 0 was significantly higher in mice receiving 4T1BR5 cells compared to mice receiving 4T1BR5-FLuc-GFP cells (p<0.0001). Mice that received 4T1BR5 cells also had significantly higher total brain tumour burden and number of brain metastases than mice that received 4T1BR5-FLuc-GFP cells (p<0.0001). Conclusions. By employing highly sensitive cellular MRI tools, we demonstrate that engineered cells did not form tumours as well as their naïve counterparts, which appear to primarily be due to a reduction in cell arrest. These results indicate that engineering cancer cells with reporter genes may alter their tropism towards particular organs and highlight another important consideration for research groups that use reporter gene imaging to track metastatic cancer cell fate in vivo.

The mechanisms that influence metastatic growth rates are poorly understood. One mechanism of interest known as concomitant tumour resistance (CTR) can be defined as the inhibition of metastasis by existing tumour mass. Conversely, the presence of a primary tumour has also been shown to increase metastatic outgrowth, termed concomitant tumour enhancement (CTE). The majority of studies evaluating CTR/CTE in preclinical models have relied on endpoint histological evaluation of tumour burden. The goal of this research was to use conventional magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging to study the impact of a primary tumour on the development of brain metastases in a syngeneic mouse model. Here, we report that the presence of a 4T1 primary tumour significantly enhances total brain tumour burden in Balb/C mice. Using in vivo BLI/MRI we could determine this was not related to differences in initial arrest or clearance of viable cells in the brain, which suggests that the presence of a primary tumour can increase the proliferative growth of brain metastases in this model. The continued application of our longitudinal cellular and molecular imaging tools will yield a better understanding of the mechanism(s) by which this physiological inhibition (CTR) and/or enhancement (CTE) occurs.