Tensor-valued diffusion encoding employs gradient waveforms that enable unique sensitivity to microstructural features of tissue, but the interpretation of signal and parameters may be confounded by diffusion-time dependence. We introduce a framework for restriction-weighted q-space trajectory imaging (ResQ) that incorporates diffusion-time effects via the restriction-weighting tensor, and we evaluate it in a longitudinal study of prostate cancer xenografts treated by external radiotherapy. We proposed a novel gradient waveform design for tensor-valued encoding with controlled restriction weighting and applied a set of four waveforms at a 9.4 T preclinical MRI system. Mice were inoculated with human prostate cancer cells (LNCaP) and assigned to groups that were untreated controls or treated by external beam irradiation. ResQ produced parameters that describe the diffusion process in terms of the mean diffusivity (D), isotropic diffusional variance (VDi), and microscopic diffusion anisotropy (VDa) as well as their diffusion-time dependence (ΔD, ΔVDi, ΔVDa). Analyses were performed to characterize parameters longitudinally and across groups. To highlight the consequences of ignoring restriction effects, we compared ResQ to analogous parameters estimated by q-space trajectory imaging (QTI). ResQ revealed clear diffusion-time dependence across all tumors, with significant longitudinal differences between treated and untreated groups, most prominent in D, ΔD, and VDi. The ResQ signal representation captured the signal dynamics, whereas QTI did not. Neglecting diffusion-time dependence in QTI led to substantial parameter bias, most notably a pronounced overestimation of microscopic diffusion anisotropy. Diffusion-time effects are non-negligible in prostate cancer and must be considered when using tensor-valued diffusion encoding. The ResQ framework enables controlled restriction weighting and improved interpretability of diffusion MRI parameters compared with approaches that ignore the effects of restriction. This provides a more principled approach for tensor-valued diffusion encoding and may enable novel imaging biomarkers that disentangle diffusivity, isotropic diffusional variance, microscopic anisotropy, and their diffusion-time dependence.

Leucine-rich repeat containing 15 (LRRC15) has emerged as an attractive biomarker and target for cancer therapy. Transforming growth factor-β (TGFβ) induces the expression of this plasma membrane protein specifically in aggressive and treatment resistant tumor cells derived from mesenchymal stem cells, with minimal expression observed in non-neoplastic tissues. We have developed a humanized monoclonal antibody, DUNP19, that specifically binds with high affinity to a phylogenetically conserved LRRC15 epitope and is rapidly internalized upon LRRC15 binding. In multiple subcutaneous and orthotopic tumor xenograft mouse models, Lutetium-177 labeled DUNP19 ([177Lu]Lu-DUNP19) enabled non-invasive imaging and molecularly precise radiotherapy to LRRC15-expressing cancer cells and murine cancer-associated fibroblasts, effectively halting tumor progression and prolonging survival with minimal toxicity. Transcriptomic analyses of [177Lu]Lu-DUNP19-treated tumors reveal a loss of pro-tumorigenic mechanisms, including a previously reported TGFβ-induced LRRC15+ signature associated with immunotherapy resistance. In a syngeneic tumor model, administration of [177Lu]Lu-DUNP19 significantly potentiated checkpoint-blockade therapy, yielding durable complete responses. Together, these results demonstrate that radio-theranostic targeting of LRRC15 with DUNP19 is a compelling precision medicine platform for image-guided diagnosis, eradication, and reprogramming of LRRC15+ tumor tissue that drives immuno-resistance and disease aggressiveness in a wide range of currently untreatable malignancies.

ABSTRACT Leucine-rich repeat containing 15 (LRRC15) has emerged as an attractive biomarker and target for cancer therapy. We have developed a humanized monoclonal antibody (mAb), DUNP19, that specifically binds to a phylogenetically conserved LRRC15 epitope and is internalized by target-expressing cancer and stromal cells. In xenograft mouse models, Lutetium-177 labeled DUNP19 ([ 177 Lu]-DUNP19) enables non-invasive imaging and precise radiotherapy to LRRC15-expressing cancer cells and murine cancer-associated fibroblasts (CAFs), halting tumor progression and prolonging survival with minimal toxicity. Transcriptomic analyses of [ 177 Lu]-DUNP19-treated tumors reveal a loss of pro-tumorigenic mechanisms, including a transforming growth factor beta (TGFβ)-driven and LRRC15+ signature associated with immunotherapy resistance. Together, these results demonstrate that radio-theranostic targeting of LRRC15 with DUNP19 is a compelling precision medicine platform for image-guided diagnosis, eradication, and reprogramming of LRRC15+ tumor tissue that drives immuno-resistance and aggressive disease. SIGNIFICANCE We introduce a pioneering LRRC15-guided radio-theranostic approach integrating clinical imaging and radioimmunotherapy. Our strategy utilizes a mAb, DUNP19, to target LRRC15-expressing cancer cells and fibroblasts, demonstrating significant tumor reduction, prolonged survival, and reversal of TGFβ-driven treatment resistance. This approach offers a promising strategy for improving outcomes in aggressive cancers.

Background Accurate tumor volume estimation is important for evaluating the response to radionuclide therapy and external beam radiotherapy as well as to other pharmaceuticals. A common method for monitoring the growth of subcutaneous tumors in pre-clinical models and assessing the treatment response is to measure the tumor length and width by external calipers to estimate its volume. This procedure relies on an assumption of a spheroidal tumor shape wherein the tumor depth equals the width and can yield considerably inaccuracies. Ultrasound imaging is a non-invasive technique that can measure all three axes of the tumor and might be an alternative to caliper measurement with potentially greater accuracy and comparable ease-of-use and throughput. Both 2D and 3D ultrasound imaging are possible, the former offering short scan times without the need for anesthesia and heating—valuable factors for longitudinal studies in large animal cohorts. Nevertheless, tumor volume estimation accuracy by 2D ultrasound imaging has seen limited investigation. In this study we have evaluated the accuracy of tumor volume estimation by caliper and 2D ultrasound with comparisons to reference measurements by magnetic resonance imaging (MRI) in a pre-clinical model of prostate cancer treated with either external beam radiotherapy, radionuclide therapy, or no treatment. Results Tumor volumes were measured longitudinally in 29 mice by caliper, ultrasound, and MRI before and after external beam radiotherapy, [177Lu]Lu-PSMA-617 radionuclide therapy, or no treatment. Caliper measurements had a marked bias, overestimating the tumor volumes by a median of 150% compared to MRI. Ultrasound measurements were markedly more accurate, with a median bias of -21% compared to MRI. Conclusion Ultrasound imaging is a reliable and accurate method for tumor volume estimation in pre-clinical models of radiotherapy, whereas caliper measurements are prone to overestimation.

We have previously investigated the biodistribution and therapy effect of a humanized monoclonal antibody targeting free prostate-specific antigen (fPSA) intended for theranostics of hormone-refractory prostate cancer. In the present study, we evaluated the off-target effect and different linear energy transfer (LET) radionuclides without the effect of PSA targeting by using an antibody with the same scaffold as previously used immunoconjugates but with random, non-specific, antigen binding region. This allows us to identify alterations generated by specific targeting and those related to passive bystander effects, such as enhanced permeability and retention (EPR). A control humanized IgG monoclonal antibody (hIgG1) and an isotype control IgG monoclonal antibody were conjugated with the chelator CHX-A"-DTPA. The immunoconjugate was radiolabeled with either Lutetium-177 ([177Lu]Lu) or Indium-111 ([111In]In). A biodistribution study in mice carrying LNCaP xenografts, was performed to evaluate the non-specific uptake of [177Lu]Lu-hIgG1 in tumors and normal organs. Further, therapy studies of [177Lu]Lu and [111In]In labeled IgG were performed in BALB/c mice carrying LNCaP xenografts. Tumor tissues of treated xenografts and control were sectioned and immunohistochemically stained for Ki67 and PSA. The highest tumor uptake for the [177Lu]Lu-hIgG1 was seen at 72 hours (7.2±2 %IA/g), when comparing the tumor uptake of the fPSA targeting antibody to the non-specific antibody, the non-specific antibody contributes to half of the tumor uptake at 72 h. The liver uptake was 3.1±0.5 %IA/g at 24 h, 2.8±0.5 %IA/g at 72 h and 1.3±0.6 %IA/g at 120 h in LNCaP xenografts, which was approximately three times lower at 24 h and two times lower at 72 h than for the antibody with preserved targeting. Immunohistochemical labeling showed a reduction of PSA expression and a reduction of Ki67 labeled cells in the [111In]In treated LNCaP tumors, compared to vehicle and [177Lu]Lu treated mice. In conclusion, we found that specific targeting might negatively influence normal organ uptake when targeting secreted antigens. Furthermore, different energy deposition i.e. linear energy transfer of a radionuclide might have diverse effects on receptor expression and cell proliferation in tumors.

Background: The humanized monoclonal antibody (mAb) hu5A10 specifically targets and internalizes prostate cancer cells by binding to prostate specific antigen (PSA). Preclinical evaluations have shown that hu5A10 is an excellent vehicle for prostate cancer (PCa) radiotheranostics. We studied the impact of different chelates and conjugation ratios on hu5A10′s target affinity, neonatal fc-receptor interaction on in vivo targeting efficacy, and possible enhanced therapeutic efficacy. Methods: In our experiment, humanized 5A10 (hu5A10) was conjugated with DOTA or DTPA at a molar ratio of 3:1, 6:1, and 12:1. Surface plasmon resonance (SPR) was used to study antigen and FcRn binding to the antibody conjugates. [111In]hu5A10 radio-immunoconjugates were administered intravenously into BALB/c mice carrying subcutaneous LNCaP xenografts. Serial Single-photon emission computed tomography (SPECT) images were obtained during the first week. Tumors were harvested and radionuclide distribution was analyzed by autoradiography along with microanatomy and immunohistochemistry. Results: As seen by SPR, the binding to PSA was clearly affected by the chelate-to-antibody ratio. Similarly, FcRn (neonatal fc-receptor) interacted less with antibodies conjugated at high ratios of chelator, which was more pronounced for DOTA conjugates. The autoradiography data indicated a higher distribution of radioactivity to the rim of the tumor for lower ratios and a more homogenous distribution at higher ratios. Mice injected with ratio 3:1 111In-DOTA-hu5A10 showed no significant difference in tumor volume when compared to mice given vehicle over a time period of 3 weeks. Mice given a similar injection of ratio 6:1 111In-DOTA-hu5A10 or 6:1 111In-DTPA-hu5A10 or 12:1 111In-DTPA-hu5A10 showed significant tumor growth retardation. Conclusions: The present study demonstrated that the radiolabeling strategy could positively modify the hu5A10′s capacity to bind PSA and complex with the FcRn-receptor, which resulted in more homogenous activity distribution in tumors and enhanced therapy efficacy.

We use multidimensional diffusion MRI to monitor longitudinal effect of external radiotherapy in human prostate cancers in mice. The measured dimensions are “diffusion time” and “shape of the b-tensor,” enabling a probe of tissue heterogeneity, microscopic anisotropy and restriction sizes. We show that the diffusivity is highly time dependent in all tumors, and that it is significantly altered by radiotherapy, already within 1-15 days of treatment. The largest effect is seen for the diffusivity and its time dependence, but the isotropic diffusional variance is also impacted. Throughout, histology is used qualitatively to provide plausible interpretations to the observations.