In vitro studies combining [¹⁷⁷Lu]Lu-DOTA-TATE with metronomic chemotherapy to improve neuroendocrine tumour therapy

Abstract

Background: Neuroendocrine tumours (NETs), the focal disease of this thesis, are a rare and biologically heterogeneous cancer originating from most organs throughout the body. NETs often display overexpression of the somatostatin receptor subtype 2 (SSTR2), the therapeutic target for second-line/beyond therapy of highly progressive NETs with systemic peptide-receptor radionuclide therapy (PRRT) using [¹⁷⁷Lu]Lu-DOTA-TATE. Treatment responses with PRRT, however, are low and thus there is great desire to improve and uncover novel treatment options to increase curative response rates for NET patients. For this thesis, the combination of [¹⁷⁷Lu]Lu-DOTA-TATE, with the DNA synthesis disrupting chemotherapies hydroxyurea, gemcitabine and triapine, was explored, in vitro, to investigate potential synergies and provide pre-clinical evidence to support recent, ongoing clinical trials investigating these combinations. The overall aims of this thesis, explored across three results chapters, are:

1. Chapter 2: Determine baseline parameters and radiobiological responses of in vitro cell models towards external beam radiotherapy (EBRT) and PRRT monotherapies, alongside assay method development.
2. Chapter 3: Assess changes in baseline parameters and radiobiological responses upon combination of EBRT and PRRT with DNA-synthesis disrupting chemotherapies.
3. Chapter 4: Compare software-calculated doses with observed biological responses from [¹⁷⁷Lu]Lu-DOTA-TATE treatments and develop a cellular dosimetry script.

Methods: The human bone osteosarcoma (U2OS) and pancreatic cancer (BON1) cell lines, also transfected for artificial SSTR2A expression (U2OS+SSTR2A and BON1+SSTR2A), were used as in vitro models for SSTR2-mediated therapy studies. Radiotherapy was given as X-ray EBRT (0-5 Gy) or [¹⁷⁷Lu]Lu-DOTA-TATE PRRT (24-hour exposure, 0.74-0.92 and 1.46-1.9 MBq), either alone (Chapter 2) or in combination with chemotherapy (Chapter 3). Specifically, metronomic concentrations of hydroxyurea (HU), 5-fluoruracil (5FU), gemcitabine (GEM) or triapine (TRI) chemotherapies, treated for 17-24 hours with a 4-hour post-chemotherapy recovery, were used. Radiobiological responses following radiotherapy and chemotherapy treatments were assessed through DNA damage (γH2AX expression, flow cytometry) and cell survival (proliferation/viability, MTT assay) studies. SSTR2A expression, cell cycle distribution and cell size alterations were also
analysed post-chemotherapy with flow cytometry. Cellular associations of [¹⁷⁷Lu]Lu-DOTA-TATE were assessed after 24-hour incubations with radioactive uptake assays, with additional cellular fractionation to determine subcellular distributions of radioactivity post-uptake. Software calculations of absorbed doses (Chapter 4) from [¹⁷⁷Lu]Lu-DOTA-TATE, performed with IDAC-Dose 2.1 and MIRDcell v3.13, were compared to equivalent EBRT doses obtained by correlating EBRT and PRRT doses to equivalent biological responses. Preliminary script development, for dose-point kernel (DPK) cellular dosimetry, was also performed in MATLAB®.

Results highlights: In Chapter 2, transfection of U2OS and BON1 cells for SSTR2A expressions were found to not induce any significant alterations in baseline cell cycle distributions or cytotoxic responses to X-ray EBRT when analysed through both γH2AX expression (p>0.9999) or metabolic viability (p=0.2681). [¹⁷⁷Lu]Lu-DOTA-TATE uptake and cytotoxicity correlated with flow cytometry analysis of SSTR2A expression, with uptake only observed in transfected U2OS+SSTR2A (0.2159 ± 0.047 Bq/cell) and BON1+SSTR2A cells (0.1112 ± 0.017 Bq/cell). Viabilities of parental cell lines displayed no significant reductions compared to controls (>90% for all added activities), unlike for U2OS+SSTR2A (0.74-0.92 MBq = 59.48±22.29%, 1.46-1.9 MBq = 40.71±14.1%) and BON1+SSTR2A cells (0.75-1.03 MBq = 45.86±10.41%). In Chapter 3, major enhancements in [¹⁷⁷Lu]Lu-DOTA-TATE therapy following chemotherapy pre-treatments were observed. In particular, [¹⁷⁷Lu]Lu-DOTA-TATE uptake (p<0.0001) increased 1.8-to-4.9-fold, with viabilities (p=0.0048) significantly decreasing to 18.85-24.45% for U2OS+SSTR2A cells compared to [¹⁷⁷Lu]Lu-DOTA-TATE monotherapy. 5FU was found to induce halted growth in U2OS/U2OS+SSTR2A cell cultures and thus omitted from combination studies. No significant enhancements, however, were observed from chemotherapy combination for either [¹⁷⁷Lu]Lu-DOTA-TATE uptake (p=0.0944) or cytotoxicity (p=0.0574) in BON1+SSTR2A cells. Alongside >80% S-phase synchronisation of in vitro cell cultures at the time of [¹⁷⁷Lu]Lu-DOTA-TATE administration, significant increases in SSTR2A expression (20.8-119.3%) and cell size (20-49.4%) were also found compared from baseline levels in both cell lines. These results partially contribute towards explaining the observed increased uptake and reduced viabilities, resulting from increased cellular capacity for internalising [¹⁷⁷Lu]Lu-DOTA-TATE. Preliminary evidence of persisted, long-term increases in radiosensitivity of cultured U2OS+SSTR2A cells towards PRRT, 3 weeks after single-dose chemotherapy, were also found. Potential SSTR2A expression enhancement was also found with targeted drug therapies, though results require further confirmation. In Chapter 4, absorbed dose calculations for [¹⁷⁷Lu]Lu-DOTA-TATE confirmed the macroscopic nature and unsuitability of IDAC-Dose 2.1 for cellular dosimetry. Trends in calculated doses from MIRDcell v3.13 for U2OS+SSTR2A cells matched experimental uptake data, with higher doses calculated for chemoradiotherapy (HU = 3.5 Gy, GEM = 7.53 Gy, TRI = 6.23 Gy) than for monotherapy (2.79 Gy). However, comparison of MIRDcell doses to biological responses observed both under- (HU = 5.14 Gy) and overestimations (GEM = 4.81 Gy, TRI = 4.86 Gy). Additionally, as MIRDcell only considered cell associated radioactivity, identical for both added activity ranges (0.74-0.92 and 1.46-1.9 MBq) due to receptor saturation observed at both activity ranges, doses could not be calculated and compared to biological responses from the higher activity range, likely having greater dose contributions from more extracellular radioactivity. Development of a cellular DPK dosimetry script, to encompass the contribution of extracellular radioactivity, was successful and executable with standard computing hardware. Validation of the script and general feasibility is now required with radionuclide-specific DPKs and with other standard computing systems.

Conclusion: Overall, the findings within this thesis present a novel and underexplored combination strategy through combining [177Lu]Lu-DOTA-TATE PRRT with metronomic levels of DNA-synthesis disrupting chemotherapy. This approach is highly amenable for translation to other cancers also treated with chemotherapy and/or radiopharmaceuticals. Whilst the findings are purely from in vitro studies, these results have great potential in informing novel clinical strategies for NETs and beyond given the widespread clinical presence of the investigated chemotherapeutics.

Date of Award	1 May 2024
Original language	English
Awarding Institution	King's College London
Supervisor	Samantha Terry (Supervisor) & Lefteris Livieratos (Supervisor)